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Sample records for 3t3 fibroblasts expressing

  1. 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 α (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 α (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

  2. TGF beta induces a sustained c-fos expression associated with stimulation or inhibition of cell growth in EL2 or NIH 3T3 fibroblasts.

    PubMed

    Liboi, E; Di Francesco, P; Gallinari, P; Testa, U; Rossi, G B; Peschle, C

    1988-02-29

    We have previously indicated that epidermal growth factor (EGF) plays a fundamental role in the proliferation control of EL2 rat fibroblast line. It is shown here that transforming growth factor beta (TGF beta) stimulates both DNA synthesis and proliferation of EL2 cells, while exerting an inhibitory effect on the growth of murine NIH-3T3 fibroblasts. We also report the effect of TGF beta and EGF on c-fos expression in EL2 cells, as compared to that of TGF beta in NIH-3T3 fibroblasts. In EL2 cells EGF induces a transient c-fos expression at both mRNA and protein level, as previously observed in NIH-3T3 fibroblasts treated with platelet-derived or fibroblast growth factor (PDGF, FGF). Conversely, TGF beta induces in EL2 cells a sustained expression of fos mRNA and protein, which are still detectable at least 24 and 7 hr after treatment respectively. In NIH-3T3 fibroblasts TGF beta causes a sustained fos RNA expression, which is not associated, however, with detectable fos protein. We conclude that in fibroblasts stimulated by mitogens c-fos expression may be differentially modulated, depending of the growth factor and the cell line. This is seemingly due to differential regulation of fos gene expression, not only at the transcriptional and/or post-transcriptional level (transient or sustained fos RNA induction by EGF or TGF beta in EL2 cells), but also at the translational level (fos protein(s) induction by TGF beta in EL2 but not NIH-3T3 fibroblasts, possibly related to the stimulatory vs inhibitory effect of this factor on the growth of the former vs the latter line).

  3. Enhanced expression of haem oxygenase-1 by nitric oxide and antiinflammatory drugs in NIH 3T3 fibroblasts.

    PubMed

    Alcaraz, M J; Habib, A; Lebret, M; Créminon, C; Lévy-Toledano, S; Maclouf, J

    2000-05-01

    1. Haem oxygenase-1 (HO-1) can exert protective effects against oxidative stress and inflammation. Fibroblasts participate in inflammatory responses where they produce high levels of prostaglandins (PGs) and nitric oxide (NO). However, little is known of the presence of HO-1 in these cells and the possible interactions among these pathways. Incubation of cells with NO donors, spermine nonoate (SPNO) and S-nitroso-N-acetylpenicillamine (SNAP), induced a dose- and time-dependent expression of HO-1 protein. 2. NO donors increased basal PGE(2) release although they reduced PGE(2) accumulated in the medium and cyclo-oxygenase (COX) activity when cells were stimulated with lipopolysaccharide (LPS). COX-2 protein was weakly induced by SPNO in basal conditions and in the presence of LPS a synergy for HO-1 and COX-2 protein expression was observed. 3. Our results indicate that reactive oxygen species participate in the inductive effect of NO donors or LPS on HO-1 expression, whereas endogenous NO production may play a role in the mechanism of the synergy exhibited by SPNO and LPS on HO-1 and COX-2 expression. In this system, zinc protoporphyrin IX did not affect nitrite levels but reduced COX activity. 4. The selective COX-2 inhibitors SC58125 and NS398 as well as the non-selective COX inhibitor, indomethacin, strongly reduced PGE(2) synthesis and showed a synergy with NO donors in HO-1 and COX-2 induction. Addition of PGE(2) had no effect, suggesting a mechanism independent of PGs formation. 5. In inflammatory conditions a number of factors could cooperate to induce HO-1 and COX-2, with a positive regulation by COX inhibitors.

  4. Fibroblast growth factor-1 stimulation of quiescent NIH 3T3 cells increases G/T mismatch-binding protein expression.

    PubMed Central

    Donohue, P J; Feng, S L; Alberts, G F; Guo, Y; Peifley, K A; Hsu, D K; Winkles, J A

    1996-01-01

    Polypeptide growth factors promote cell-cycle progression in part by the transcriptional activation of a diverse group of specific genes. We have used an mRNA differential-display approach to identify several fibroblast growth factor (FGF)-1 (acidic FGF)-inducible genes in NIH 3T3 cells. Here we report that one of these genes, called FGF-regulated (FR)-3, is predicted to encode G/T mismatch-binding protein (GTBP), a component of the mammalian DNA mismatch correction system. The murine GTBP gene is transiently expressed after FGF-1 or calf serum treatment, with maximal mRNA levels detected at 12 and 18 h post-stimulation. FGF-1-stimulated NIH 3T3 cells also express an increased amount of GTBP as determined by immunoblot analysis. These results indicate that elevated levels of GTBP may be required during the DNA synthesis phase of the cell cycle for efficient G/T mismatch recognition and repair. PMID:8870641

  5. 3T3 fibroblasts transfected with a cDNA for mitochondrial aspartate aminotransferase express plasma membrane fatty acid-binding protein and saturable fatty acid uptake.

    PubMed Central

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

    1995-01-01

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

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

    PubMed

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

    2014-04-04

    Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA1-LPA6) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA1 inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA5 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 LPA1 and LPA5 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 LPA5 may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA1.

  7. 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}.

  8. Expression of discoidin domain receptor 2 (DDR2) extracellular domain in pichia pastoris and functional analysis in synovial fibroblasts and NIT3T3 cells.

    PubMed

    Zhang, Wei; Ding, Tianbing; Zhang, Jian; Su, Jin; Li, Fuyang; Liu, Xinping; Ma, Wenyu; Yao, Libo

    2006-10-01

    Discoidin domain receptor 2 (DDR2) is a kind of protein tyrosine kinases associated with cell proliferation and tumor metastasis, and collagen, identified as a ligand for DDR2, up-regulates matrix metallloproteinase 1 (MMP-1) and MMP-2 expression in cellular matrix. To investigate the roles of DDR2 in destruction of cartilage in rheumatoid arthritis (RA) and tumor metastasis, we tried to express extracellular domain of DDR2 fused with a His tag to increase protein solubility and facilitate purification (without signal peptide and transmembrane domain, designated DR) in Pichia pastoris, purify the expressed protein, and characterize its function, for purpose of future application as a specific DDR2 antagonist. Two clones of relative high expression of His-DR were obtained. After purification by a Ni-NTA (nitric-tri-acetic acid) chromatographic column, soluble fused His-DR over 90% purity were obtained. Competitive binding inhibition assay demonstrated that expressed His-DR could block the binding of DDR2 and natural DDR2 receptors on NIT3T3 and synovial cell surfaces. Results of RT-PCR, Western blotting, and gelatinase zymography showed that His-DR was capable of inhibiting MMP-1 and MMP-2 secretion from NIT3T3 cells and RA synoviocytes stimulated by collagen II. For MMP-1, the inhibitory effect was displayed at the levels of mRNA and protein, whereas for MMP-2 it was demonstrated at the level of protein physiological activity. All these findings suggested that the fused expressed His-DR inhibited the activity of natural DDR2, and relevant MMP-1 and MMP-2 expression in synoviocytes and NIH3T3 cells provoked by collagen II.

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

    PubMed

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

    2009-12-01

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

  10. Study of lactoferrin gene expression in human and mouse adipose tissue, human preadipocytes and mouse 3T3-L1 fibroblasts. Association with adipogenic and inflammatory markers.

    PubMed

    Moreno-Navarrete, José María; Serrano, Marta; Sabater, Mònica; Ortega, Francisco; Serino, Matteo; Pueyo, Neus; Luche, Elodie; Waget, Aurelie; Rodriguez-Hermosa, José Ignacio; Ricart, Wifredo; Burcelin, Remy; Fernández-Real, José Manuel

    2013-07-01

    Lactoferrin is considered an epithelial protein present in different gland secretions. Administration of exogenous lactoferrin is also known to modulate adipogenesis and insulin action in human adipocytes. Here, we aimed to investigate lactoferrin gene expression (real-time polymerase chain reaction) and protein (enzyme-linked immunosorbent assay) levels in human (n=143) and mice adipose tissue samples, in adipose tissue fractions and during human preadipocyte and 3T3-L1 cell line differentiation, evaluating the effects of inducers (rosiglitazone) and disruptors (inflammatory factors) of adipocyte differentiation. Lactoferrin (LTF) gene and protein were detectable at relatively high levels in whole adipose tissue and isolated adipocytes in direct association with low-density lipoprotein-related protein 1 (LRP1, its putative receptor). Obese subjects with type 2 diabetes and increased triglycerides had the lowest levels of LTF gene expression in subcutaneous adipose tissue. Specifically, LTF gene expression was significantly increased in adipocytes, mainly from lean subjects, increasing during differentiation in parallel to adipogenic genes and gene markers of lipid droplets. The induction or disruption of adipogenesis led to concomitant changes (increase and decrease, respectively) of lactoferrin levels during adipocyte differentiation also in parallel to gene markers of adipogenesis and lipid droplet development. The administration of lactoferrin led to autopotentiated increased expression of the LTF gene. The decreased lactoferrin mRNA levels in association with obesity and diabetes were replicated in mice adipose tissue. In conclusion, this is the first observation, to our knowledge, of lactoferrin gene expression in whole adipose tissue and isolated adipocytes, increasing during adipogenesis and suggesting a possible contribution in adipose tissue physiology through LRP1.

  11. 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.

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

    SciTech Connect

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

    2010-02-19

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

  13. 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

  14. Interferon inhibits the conversion of 3T3-L1 mouse fibroblasts into adipocytes.

    PubMed Central

    Keay, S; Grossberg, S E

    1980-01-01

    Confluent Swiss mouse 3T3-L1 fibroblasts slowly differentiate functionally and morphologically into adipocytes, a conversion hastened by insulin. The cells are sensitive (although less than L929 cells) to the antiviral action of mouse fibroblast interferons but not to interferons from heterologous species (human and chicken). Cultures stimulated with insulin in the presence of partially purified or electrophoretically pure mouse interferons have a much lower percentage of cells accumulating lipid than do insulin-treated control cultures. Interferon-treated cell cultures also contain much less triglyceride, cholesterol, and cholesterol esters than do replicate control cultures stimulated by insulin to differentiate. Increased de novo lipid biosynthesis that occurs during differentiation is inhibited, as determined by incorporation of [14C]acetate into lipids extractable by the Folch method. This incorporation is a sensitive bioassay of the antidifferentiation effect of interferon; less than 1 antiviral unit is inhibitory. Variously inactivated or mock interferon preparations as well as interferons from several heterologous species fail to inhibit 3T3-L1 adipocyte conversion. Interferon is inhibitory even when applied as long as 3 days after insulin stimulation. The effect of interferon does not appear to depend upon its competition with insulin for cell surface receptors. Because interferon can alter the program of events involved in conversion of 3T3-L1 fibroblasts into adipose cells, it may be able to affect the regulation of eukaryotic cell differentiation. Images PMID:6159626

  15. 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

  16. 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

  17. Strategies of NF-κB signaling modulation by ectromelia virus in BALB/3T3 murine fibroblasts.

    PubMed

    Struzik, Justyna; Szulc-Dąbrowska, Lidia; Winnicka, Anna; Niemiałtowski, Marek

    2015-10-01

    Nuclear factor κB (NF-κB) is a pleiotropic transcription factor that regulates the expression of immune response genes. NF-κB 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-κB signaling in murine BALB/3T3 fibroblasts. Activation of NF-κB via tumor necrosis factor (TNF) receptor 1 (TNFR1) in these cells induces proinflammatory cytokine secretion. We show that ECTV does not recruit NF-κB to viral factories or induce NF-κB nuclear translocation in BALB/3T3 cells. Additionally, ECTV counteracts TNF-α-induced p65 NF-κB nuclear translocation during the course of infection. Inhibition of TNF-α-induced p65 nuclear translocation was also observed in neighboring cells that underwent fusion with ECTV-infected cells. ECTV inhibits the key step of NF-κB activation, i.e. Ser32 phosphorylation and degradation of inhibitor κBα (IκBα) induced by TNF-α. We also observed that ECTV prevents TNF-α-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.

  18. Inhibitory effects of LPA1 on cell motile activities stimulated by hydrogen peroxide and 2,3-dimethoxy-1,4-naphthoquinone in fibroblast 3T3 cells.

    PubMed

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

    2013-11-08

    Reactive oxygen species (ROS) are known to mediate a variety of biological responses, including cell motility. Recently, we indicated that lysophosphatidic acid (LPA) receptor-3 (LPA3) increased cell motile activity stimulated by hydrogen peroxide. In the present study, we assessed the role of LPA1 in the cell motile activity mediated by ROS in mouse fibroblast 3T3 cells. 3T3 cells were treated with hydrogen peroxide and 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) at concentrations of 0.1 and 1 μM for 48 h. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3 cells treated with hydrogen peroxide and DMNQ were significantly higher than those of untreated cells. 3T3 cells treated with hydrogen peroxide and DMNQ showed elevated expression levels of the Lpar3 gene, but not the Lpar1 and Lpar2 genes. To investigate the effects of LPA1 on the cell motile activity induced by hydrogen peroxide and DMNQ, Lpar1-overexpressing (3T3-a1) cells were generated from 3T3 cells and treated with hydrogen peroxide and DMNQ. The cell motile activities stimulated by hydrogen peroxide and DMNQ were markedly suppressed in 3T3-a1 cells. These results suggest that LPA signaling via LPA1 inhibits the cell motile activities stimulated by hydrogen peroxide and DMNQ in 3T3 cells.

  19. Lipid droplet changes in proliferating and quiescent 3T3 fibroblasts.

    PubMed

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

    2008-05-01

    Lipid droplets (LDs) are fat-storing organelles present in virtually all eukaryotic cells and involved in many aspects of cell biology related to lipid metabolism and cholesterol homeostasis. In this study, we investigated the presence of LDs in proliferating and quiescent (contact-inhibited) 3T3 fibroblasts to verify a correlation with cell growth. LDs were characterized by Nile red staining, positivity to adipophilin and negativity to perilipin. LDs were numerous in proliferating cells, but very few in quiescent cells. However, the fraction of quiescent cells, which resumed proliferation after scratch-wound assay, also resumed the formation of LDs. In proliferating cells, the number of LDs correlated with the DNA content, suggesting a continuous accumulation of LDs during cell growth. These findings were supported by biochemical data showing much higher rates of cholesterol esterification and triglyceride synthesis in proliferating cells. Both filipin staining and the fluorescent cholesterol analog dehydroergosterol revealed the presence of an intense traffic of free cholesterol, mediated by acidic vesicles, in proliferating cells. Nile red ratiometric measurements revealed a different lipid composition of LDs in proliferating and quiescent cells. Changes in the number and composition of LDs were also found in growing cells treated with inhibitors of cholesterol esterification (Sandoz 58-035), endosomal cholesterol efflux (U18666A) and V-ATPase (bafilomycin-A1).

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

    SciTech Connect

    Kohler, C.; Petersen, R.

    1986-03-01

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

  1. 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

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

    PubMed Central

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

    1991-01-01

    Culture systems designed to maintain or develop human mast cells have proved difficult, yet these systems would provide valuable resources for future investigations of human mast cell biology. Cocultures of either isolated mature human lung mast cells (Levi-Schaffer et al., J Immunol 1987, 139:494-500) or human cord blood mononuclear cells (Furitsu, Proc Natl Acad Sci USA 1989, 86:10039-10043) with 3T3 embryonic mouse skin fibroblasts have implicated fibroblasts as an important factor in the successful maintenance and development of human mast cells in vitro. The authors cultured isolated, mature human lung mast cells either with or without 3T3 cells for 1 month and examined their ultrastructural phenotype. Mast cell viability in each circumstance was equivalent, but mast cell yield was improved in the presence of 3T3 cells. The ultrastructural phenotype was identical in both culture systems. Mast cells were shown to maintain the phenotype of their in vivo lung counterparts (ie, scroll granules predominanted, and numerous lipid bodies were present). This ultrastructural phenotype differs from that of mast cells that develop in cocultures of human cord blood cells and 3T3 cells, where developing mast cells with crystalline granules and few lipid bodies prevail, a phenotype much like that of human skin mast cells in vivo (Furitsu, Proc Natl Acad Sci USA 1989, 86:10039-10043). Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:1750506

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

    SciTech Connect

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

    1989-04-01

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

  4. Comparative Culturing of 3T3 Swiss J2 Mouse Embryo Fibroblasts on Modified Chitosan Matrices.

    PubMed

    Alekhin, A I; Gaenko, G P

    2016-07-01

    Comparative culturing of mouse embryo fibroblasts on chitosan matrices and culture plastic was carried out. During the first 2 h of culturing (lag phase), cell adhesion to chitosan and chitosan-gelatin matrices was 20-30% higher than adhesion to culture plastic (control). During the stationary phase, 80% cells adhered to chitosan matrices (vs. 60% in the control). Cell culturing on chitosan matrices was carried out without medium replacement with fresh portions. The cells remained viable within 5 days of culturing. Cell death phase was observed on day 6 of culturing on chitosan matrices: cell adhesion dropped to 50%. Culturing on culture plastic was carried out with daily medium replacement with a fresh portion. Cell death phase (50% decrease in the number of adherent cell) under these condition was observed on day 5. It seems that the observed effect was a result of contact interactions of cell integrins and chitosan ligands, modulation of transmembrane signal, eventually modifying the expression of cell genes. This effect can be required in regenerative medicine for production of primary cell culture.

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

    SciTech Connect

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

    1986-03-05

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

  6. Uncoupling of 3T3-L1 gene expression from lipid accumulation during adipogenesis.

    PubMed

    Temple, Karla A; Basko, Xheni; Allison, Margaret B; Brady, Matthew J

    2007-02-06

    Adipocyte differentiation comprises altered gene expression and increased triglyceride storage. To investigate the interdependency of these two events, 3T3-L1 cells were differentiated in the presence of glucose or pyruvate. All adipocytic proteins examined were similarly increased between the two conditions. In contrast, 3T3-L1 adipocytes differentiated with glucose exhibited significant lipid accumulation, which was largely suppressed in the presence of pyruvate. Subsequent addition of glucose to the latter cells restored lipid accumulation and acute rates of insulin-stimulated lipogenesis. These data indicate that extracellular energy is required for induction of adipocytic proteins, while only glucose sustained the parallel increase in triglyceride storage.

  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. Cloning and Expression of CD19, a Human B-Cell Marker in NIH-3T3 Cell Line

    PubMed Central

    Abbasi-Kenarsari, Hajar; Shafaghat, Farzaneh; Baradaran, Behzad; Movassaghpour, Ali Akbar; Shanehbandi, Dariush; Kazemi, Tohid

    2015-01-01

    Background CD19 is a pan B cell marker that is recognized as an attractive target for antibody-based therapy of B-cell disorders including autoimmune disease and hematological malignancies. The object of this study was to stably express the human CD19 antigen in the murine NIH-3T3 cell line aimed to be used as an immunogen in our future study. Methods Total RNA was extracted from Raji cells in which high expression of CD19 was confirmed by flow cytometry. Synthesized cDNA was used for CD19 gene amplification by conventional PCR method using Pfu DNA polymerase. PCR product was ligated to pGEM-T Easy vector and ligation mixture was transformed to DH5α competent bacteria. After blue/white selection, one positive white colony was subjected to plasmid extraction and direct sequencing. Then, CD19 cDNA was sub-cloned into pCMV6-Neo expression vector by double digestion using KpnI and HindIII enzymes. NIH-3T3 mouse fibroblast cell line was subsequently transfected by the construct using Jet-PEI transfection reagent. After 48 hours, surface expression of CD19 was confirmed by flow cytometry and stably transfected cells were selected by G418 antibiotic. Results Amplification of CD19 cDNA gave rise to 1701 bp amplicon confirmed by alignment to reference sequence in NCBI database. Flow cytometric analysis showed successful transient and stable expression of CD19 on NIH-3T3 cells (29 and 93%, respectively). Conclusion Stable cell surface expression of human CD19 antigen in a murine NIH-3T3 cell line may develop a proper immunogene which raises specific anti-CD19 antibody production in the mice immunized sera. PMID:25926951

  9. Malignant transformation induced by incorporated radionuclides in BALB/3T3 mouse embryo fibroblasts.

    PubMed Central

    LeMotte, P K; Adelstein, S J; Little, J B

    1982-01-01

    The induction of lethality and malignant transformation by 5-[125I]iododeoxyuridine and [3H]thymidine incorporated into cellular DNA and by x-irradiation was studied in vitro in BALB/3T3 cells. Under these conditions, 125I radiation is highly localized to small regions of the DNA at the site of each decay and produces DNA double-strand breaks with high efficiency. Incorporated 125I was found to be 12-16 times as lethal per decay as incorporated 3H. For the induction of malignant transformation, however, 125I was approximately 25 times as effective per decay as 3H. When the frequencies of transformation induced at various levels of survival by 125I, 3H, and x-rays were compared, lethality was found to correlate closely with transformation at doses that yielded significant cell killing. An exception occurred at low doses, where 125I appeared much more efficient than x-irradiation in inducing transformation; transformation frequencies equal to those induced by 3-5 Gy of x-rays resulted from 125I exposures that yielded little or no cell killing. PMID:6961448

  10. Mechanisms of vitamin K transport and metabolism in Swiss 3T3 mouse fibroblasts

    SciTech Connect

    Canfield, L.M.; Townsend, A.F.; Hibbs, D.B.

    1986-03-01

    Transport of vitamin K into isolated fibroblasts was followed using /sup 3/H vitamin K/sub 1/. The initial rate is saturable by 5 min. at 25..mu..M vitamin K with a Km(app) of 10..mu..M and V/sub max/ of 50 pmols/min/10/sup 6/ cells. Kinetics of uptake are biphasic with a second slower rate ensuing after 10 minutes. Insensitivity of the initial rate of uptake to FCCP or ouabain indicates an ATP-independent transport mechanism. Specificity of transport is shown by competition of uptake of /sup 3/H vitamin K by unlabelled vitamin and strong (>90%) inhibition of the initial rate by equimolar concentrations of the vitamin K analog, Chloro-K. In addition, following uptake, both vitamins K/sub 1/ and K/sub 2/ are metabolized to their respective epoxides. Vitamin K/sub 1/ epoxide is also transported into fibroblasts and metabolized to the parent quinone in a Warfarin-sensitive reaction. Following alkaline hydrolysis of isolated intracellular protein, the vitamin K-dependent amino acid, gamma carboxyglutamic acid (gla) was detected. It is concluded that vitamin K is specifically transported into fibroblasts and metabolized via the classical pathway described in liver with the concomitant production of vitamin K-dependent proteins.

  11. Immunohistochemical evidence for an association of ribosomes with microfilaments in 3T3 fibroblasts.

    PubMed

    Hesketh, J E; Horne, Z; Campbell, G P

    1991-02-01

    Ribosome distribution in cultured fibroblasts was investigated immunohistochemically using antibodies which recognize the 60S ribosomal subunit. After treatment of cells with buffer containing 25mM KCl and 0.05% Nonidet-P40 immunostained material was present in punctate patterns and linear arrays consistent with some ribosomes being associated with the cytoskeleton. Treatment of the cells with 130mM KCl caused loss of both the beaded lines of immunostaining and micro-filaments. Double immunostaining showed ribosomes to be closely associated with microfilaments.

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

    SciTech Connect

    Takahashi, Nobuhiko; 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-γ 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-α and interleukin-1β 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-γ in 3T3-L1 adipocytes. Activation of peroxisome proliferator-activated receptor-γ 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. 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.

  14. Menaquinone-7 regulates gene expression in osteoblastic MC3T3E1 cells.

    PubMed

    Katsuyama, Hironobu; Saijoh, Kiyofumi; Otsuki, Takemi; Tomita, Masafumi; Fukunaga, Masao; Sunami, Shigeo

    2007-02-01

    Previous study has shown that the vitamin K2 analog menaquinone-7 (MK-7) induces expression of the osteoblast-specific genes osteocalcin, osteoprotegerin, receptor activator of NFkappaB, and its ligand. Since MK-7 may also regulate osteoblast cell function, we examined the expression of osteoblast genes regulated by MK-7 administration. Differences between gene expression in control and MK-7-administered MC3T3E1 cells were analyzed using the suppression subtractive hybridization method. After 24 h of MK-7 administration, genes upregulated by MK-7 included tenascin C and BMP2. Genes downregulated by MK-7 administration included biglycan and butyrophilin. Real-time PCR showed a marked increase in tenascin C. When the protein level was examined using Western blot analysis, tenascin C was higher in MK-7-administered cells than in control cells. These results indicated that MK-7 affected the cellular function of osteoblastic MC3T3E1 cells. Considering BMP2 mRNA expression was higher in MK-7-administered cells than in control cells, the effect of MK-7 administration on the signal transduction system was examined. Western blot analysis showed that cells administered MK-7 displayed a higher phosphorylated Smad1 level than control cells. Because MC3T3E1 cells have a nuclear binding receptor for MK-7, this result might indicate an indirect effect of MK-7 through BMP2 production.

  15. Nanofiber Alignment Regulates NIH3T3 Cell Orientation and Cytoskeletal Gene Expression on Electrospun PCL+Gelatin Nanofibers

    PubMed Central

    Fee, Timothy; Surianarayanan, Swetha; Downs, Crawford; Zhou, Yong; Berry, Joel

    2016-01-01

    To examine the influence of substrate topology on the behavior of fibroblasts, tissue engineering scaffolds were electrospun from polycaprolactone (PCL) and a blend of PCL and gelatin (PCL+Gel) to produce matrices with both random and aligned nanofibrous orientations. The addition of gelatin to the scaffold was shown to increase the hydrophilicity of the PCL matrix and to increase the proliferation of NIH3T3 cells compared to scaffolds of PCL alone. The orientation of nanofibers within the matrix did not have an effect on the proliferation of adherent cells, but cells on aligned substrates were shown to elongate and align parallel to the direction of substrate fiber alignment. A microarray of cyotoskeleton regulators was probed to examine differences in gene expression between cells grown on an aligned and randomly oriented substrates. It was found that transcriptional expression of eight genes was statistically different between the two conditions, with all of them being upregulated in the aligned condition. The proteins encoded by these genes are linked to production and polymerization of actin microfilaments, as well as focal adhesion assembly. Taken together, the data indicates NIH3T3 fibroblasts on aligned substrates align themselves parallel with their substrate and increase production of actin and focal adhesion related genes. PMID:27196306

  16. 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.

  17. Treatment with LPS plus INF-γ induces the expression and function of muscarinic acetylcholine receptors, modulating NIH3T3 cell proliferation: participation of NOS and COX

    PubMed Central

    Español, A J; Maddaleno, M O; Lombardi, M G; Cella, M; Martínez Pulido, P; Sales, M E

    2014-01-01

    Background and Purpose LPS and IFN-γ are potent stimuli of inflammation, a process in which fibroblasts are frequently involved. We analysed the effect of treatment with LPS plus IFN-γ on the expression and function of muscarinic acetylcholine receptors in NIH3T3 fibroblasts with regards to proliferation of these cells. We also investigated the participation of NOS and COX, and the role of NF-κB in this process. Experimental Approach NIH3T3 cells were treated with LPS (10 ng·mL−1) plus IFN-γ (0.5 ng·mL−1) for 72 h (iNIH3T3 cells). Cell proliferation was evaluated with MTT and protein expression by Western blot analysis. NOS and COX activities were measured by the Griess method and radioimmunoassay respectively. Key Results The cholinoceptor agonist carbachol was more effective at stimulating proliferation in iNIH3T3 than in NIH3T3 cells, probably due to the de novo induction of M3 and M5 muscarinic receptors independently of NF-κB activation. iNIH3T3 cells produced higher amounts of NO and PGE2 than NIH3T3 cells, concomitantly with an up-regulation of NOS1 and COX-2, and with the de novo induction of NOS2/3 in inflamed cells. We also found a positive feedback between NOS and COX that could potentiate inflammation. Conclusions and Implications Inflammation induced the expression of muscarinic receptors and, therefore,stimulated carbachol-induced proliferation of fibroblasts. Inflammation also up-regulated the expression of NOS and COX-2, thus potentiating the effect of carbachol on NO and PGE2 production. A positive crosstalk between NOS and COX triggered by carbachol in inflamed cells points to muscarinic receptors as potential therapeutic targets in inflammation. PMID:24990429

  18. Retinoic acid inhibits inducible nitric oxide synthase expression in 3T3-L1 adipocytes.

    PubMed

    Yang, Jeong-Yeh; Koo, Bon-Sun; Kang, Mi-Kyung; Rho, Hye-Won; Sohn, Hee-Sook; Jhee, Eun-Chung; Park, Jin-Woo

    2002-11-30

    The present study was undertaken to explore whether retinoids, which are known to have immunomodulatory actions, could attenuate tumor necrosis factor-alpha (TNF)-stimulated inducible nitric oxide synthase (iNOS) expression in 3T3-L1 adipocytes. Adipocytes incubated with TNF induced dose- and time-dependent accumulation of nitrite in the culture medium through the iNOS induction as confirmed by Western blotting. Treatment of cells with TNF in the presence of all-trans-retinoic acid (RA) significantly decreased their ability to produce nitrite and iNOS induction. Both 13-cis- and all- trans-RA-induced suppression was dose-dependent, and all-trans-RA was somewhat potent than 13-cis-RA. The inhibitory effect of RA on TNF-induced iNOS induction was reversible, completely recovered after 2 days, and was exerted through the inhibition of NF-kappaB activation. TNF also suppressed the lipoprotein lipase (LPL) activity of 3T3-L1 adipocytes. RA could not reverse the TNF- induced LPL suppression at RA levels causing near complete inhibition of the TNF-induced NO production. These results indicate that RAs attenuate iNOS expression reversibly in TNF-stimulated 3T3-L1 adipocytes, and that the TNF-induced LPL suppression is not the result of NO overproduction.

  19. Erythropoietin and interleukin-1beta modulate nitrite production in a Swiss 3T3 cell model of rheumatoid synovial fibroblasts.

    PubMed

    Baig, S; Patel, Y; Coussons, P; Grant, R

    2002-11-01

    Erythropoietin (EPO), a haemopoietic growth factor and a primary regulator of erythropoiesis, is widely used to treat anaemia in various chronic complications of rheumatoid arthritis (RA). Fibroblast-like cells, found in the pannus tissue of joints, are thought to contribute to the inflammatory pathology of RA. Thus for the current study we investigated the effects of recombinant human EPO (rHuEPO) on NO metabolism, using an interleukin-1beta (IL-1beta)-stimulated Swiss 3T3 fibroblast monolayer as a model for fibroblast activity in RA. The results show that, over 3 days, both alone and in combination with the pro-inflammatory cytokine IL-1beta (10 ng/ml), rHuEPO (25 micro-units/ml) induced significant production of nitrite in cell culture supernatants. This is an indicator of NO production by nitric oxide synthase (NOS), which is a well-documented mediator of metalloproteinase-mediated tissue remodelling in RA. It therefore appears that, through modulation of NOS-dependent NO production, rHuEPO may influence remodelling of connective tissue in RA, independently of its established erythropoietic role.

  20. The aporphine alkaloid boldine induces adiponectin expression and regulation in 3T3-L1 cells.

    PubMed

    Yu, Bangning; Cook, Carla; Santanam, Nalini

    2009-10-01

    Adiponectin is an adipokine secreted by differentiated adipocytes. Clinical studies suggest a negative correlation between oxidative stress and adiponectin levels in patients with metabolic syndrome or cardiovascular disease. Natural compounds that can prevent oxidative stress mediated inhibition of adiponectin may be potentially therapeutic. Boldine, an aporphine alkaloid abundant in the medicinal plant Peumus boldus, is a powerful antioxidant. The current study demonstrates the effects of boldine on the expression of adiponectin and its regulators, CCAAT/enhancer binding protein-alpha (C/EBPalpha) and peroxisome proliferator-activated receptor (PPAR)-gamma, in 3T3-L1 cells. Differentiated 3T3-L1 adipocytes were exposed to either hydrogen peroxide (H(2)O(2)) (100 microM) or tumor necrosis factor-alpha (TNFalpha) (1 ng/mL) for 24 hours in the presence or absence of increasing concentrations of boldine (5-100 microM). Quantitative polymerase chain reaction showed that both the oxidants decreased the mRNA levels of adiponectin, PPARgamma, and C/EBPalpha to half of the control levels. Boldine, at all concentrations, counteracted the inhibitory effect of H(2)O(2) or TNFalpha and increased the expression of adiponectin and its regulators. The effect of boldine on adiponectin expression was biphasic, with the lower concentrations (5-25 microM) having a larger inductive effect compared to higher concentrations (50-100 microM). Boldine treatment alone in the absence of H(2)O(2) or TNFalpha was also able to induce adiponectin at the inductive phase of adipogenesis. Peroxisome proliferator response element-luciferase promoter transactivity analysis showed that boldine interacts with the PPAR response element and could potentially modulate PPAR responsive genes. Our results indicate that boldine is able to modulate the expression of adiponectin and its regulators in 3T3-L1 cells and has the potential to be beneficial in obesity-related cardiovascular disease.

  1. The Aporphine Alkaloid Boldine Induces Adiponectin Expression and Regulation in 3T3-L1 Cells

    PubMed Central

    Yu, Bangning; Cook, Carla

    2009-01-01

    Abstract Adiponectin is an adipokine secreted by differentiated adipocytes. Clinical studies suggest a negative correlation between oxidative stress and adiponectin levels in patients with metabolic syndrome or cardiovascular disease. Natural compounds that can prevent oxidative stress mediated inhibition of adiponectin may be potentially therapeutic. Boldine, an aporphine alkaloid abundant in the medicinal plant Peumus boldus, is a powerful antioxidant. The current study demonstrates the effects of boldine on the expression of adiponectin and its regulators, CCAAT/enhancer binding protein-α (C/EBPα) and peroxisome proliferator-activated receptor (PPAR)-γ, in 3T3-L1 cells. Differentiated 3T3-L1 adipocytes were exposed to either hydrogen peroxide (H2O2) (100 μM) or tumor necrosis factor-α (TNFα) (1 ng/mL) for 24 hours in the presence or absence of increasing concentrations of boldine (5–100 μM). Quantitative polymerase chain reaction showed that both the oxidants decreased the mRNA levels of adiponectin, PPARγ, and C/EBPα to half of the control levels. Boldine, at all concentrations, counteracted the inhibitory effect of H2O2 or TNFα and increased the expression of adiponectin and its regulators. The effect of boldine on adiponectin expression was biphasic, with the lower concentrations (5–25 μM) having a larger inductive effect compared to higher concentrations (50–100 μM). Boldine treatment alone in the absence of H2O2 or TNFα was also able to induce adiponectin at the inductive phase of adipogenesis. Peroxisome proliferator response element-luciferase promoter transactivity analysis showed that boldine interacts with the PPAR response element and could potentially modulate PPAR responsive genes. Our results indicate that boldine is able to modulate the expression of adiponectin and its regulators in 3T3-L1 cells and has the potential to be beneficial in obesity-related cardiovascular disease. PMID:19857072

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

    SciTech Connect

    Jozaki, K.; Kuriu, A.; Hirota, S.; Onoue, H.; Ebi, Y.; Adachi, S.; Ma, J.Y.; Tarui, S.; Kitamura, Y. )

    1991-03-01

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

  3. Dynamics of Actin Stress Fibers and Focal Adhesions during Slow Migration in Swiss 3T3 Fibroblasts: Intracellular Mechanism of Cell Turning

    PubMed Central

    Miyoshi, Hiromi; Miura, Takuya; Tanaka, Hiroto; Tsubota, Ken-ichi; Liu, Hao

    2016-01-01

    To understand the mechanism regulating the spontaneous change in polarity that leads to cell turning, we quantitatively analyzed the dynamics of focal adhesions (FAs) coupling with the self-assembling actin cytoskeletal structure in Swiss 3T3 fibroblasts. Fluorescent images were acquired from cells expressing GFP-actin and RFP-zyxin by laser confocal microscopy. On the basis of the maximum area, duration, and relocation distance of FAs extracted from the RFP-zyxin images, the cells could be divided into 3 regions: the front region, intermediate lateral region, and rear region. In the intermediate lateral region, FAs appeared close to the leading edge and were stabilized gradually as its area increased. Simultaneously, bundled actin stress fibers (SFs) were observed vertically from the positions of these FAs, and they connected to the other SFs parallel to the leading edge. Finally, these connecting SFs fused to form a single SF with matured FAs at both ends. This change in SF organization with cell retraction in the first cycle of migration followed by a newly formed protrusion in the next cycle is assumed to lead to cell turning in migrating Swiss 3T3 fibroblasts. PMID:28119928

  4. Roughness threshold for cell attachment and proliferation on plasma micro-nanotextured polymeric surfaces: the case of primary human skin fibroblasts and mouse immortalized 3T3 fibroblasts

    NASA Astrophysics Data System (ADS)

    Bourkoula, A.; Constantoudis, V.; Kontziampasis, D.; Petrou, P. S.; Kakabakos, S. E.; Tserepi, A.; Gogolides, E.

    2016-08-01

    Poly(methyl methacrylate) surfaces have been micro-nanotextured in oxygen plasmas with increasing ion energy, leading to micro-nanotopography characterized by increased root mean square roughness, correlation length and fractal dimension. Primary human skin fibroblasts and mouse immortalized 3T3 fibroblasts were cultured on these surfaces and the number of adhering cells, their proliferation rate and morphology (cytoplasm and nucleus area) were evaluated as a function of roughness height, correlation length, and fractal dimension. A roughness threshold behavior was observed for both types of cells leading to dramatic cell number decrease above this threshold, which is almost similar for the two types of cells, despite their differences in size and stiffness. The results are discussed based on two theoretical models, which are reconciled and unified when the elastic moduli and the size of the cells are taken into account.

  5. Regulation of the beta-adrenergic receptor-adenylate cyclase complex of 3T3-L1 fibroblasts by sodium butyrate

    SciTech Connect

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

    1986-05-01

    Mouse 3T3-L1 fibroblasts contain beta-adrenergic receptors (BAR), predominantly of the B/sub 1/ subtype. Incubation of these cells with 2-10 mM sodium butyrate (SB) for 24-48 hr results in a switch in the BAR subtype from B/sub 1/ to B/sub 2/ and promotes a 1.5 to 2.5 fold increase in total BAR number. Other short chain acids were not as effective as SB in promoting changes in BAR. BAR were assayed in membranes prepared from the 3T3-L1 cells using the radiolabeled antagonist (/sup 125/I)-cyanopindolol and the B/sub 2/ selective antagonist ICI 118.551. BAR subtype switch was confirmed functionally by measuring cellular cAMP accumulation in response to agonists. The structure and amount of the alpha subunits of the guanine nucleotide regulatory proteins N/sub s/ and N/sub i/ were determined by ADP-ribosylation using /sup 32/P-NAD and either cholera toxin or pertussis toxin for labeling of the respective subunits. Preincubation of cells with 5 mM SB for 48 hr resulted in a 2-3 fold increase in the labeling of the alpha subunits of both N/sub s/ and N/sub i/. A protein of M/sub r/ = 44,000 showed enhanced labeling by cholera toxin following SB treatment of the cells. These data indicate SB concomitantly regulates expression of BAR subtype and components of the adenylate cyclase in 3T3-L1 cells.

  6. 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 PPARγ 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 PPARγ expression. Mechanistically, fisetin facilitated Sirt1-mediated deacetylation of PPARγ and FoxO1, and enhanced the association of Sirt1 with the PPARγ promoter, leading to suppression of PPARγ transcriptional activity, thereby repressing adipogenesis. Lowering Sirt1 levels reversed the effects of fisetin on deacetylation of PPARγ and increased PPARγ transactivation. Collectively, our results suggest the effects of fisetin in increasing Sirt1 expression and in epigenetic control of early adipogenesis.

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

    PubMed Central

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

    2015-01-01

    P16 is an acidic phosphoprotein important in both sea urchin embryonic spicule development and transient mineralization during embryogenesis, and 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 β-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. PMID:26581835

  8. Triiodothyronine modulates the expression of leptin and adiponectin in 3T3-L1 adipocytes

    PubMed Central

    de Oliveira, Miriane; Síbio, Maria Teresa De; Olimpio, Regiane Marques Castro; Moretto, Fernanda Cristina Fontes; Luvizotto, Renata de Azevedo Melo; Nogueira, Celia Regina

    2015-01-01

    Objective To study the effect of different doses of triiodothyronine on gene expression of the adipokines leptin and adiponectin, at different times, and to evaluate the difference in expression between the two adipokines in each group. Methods 3T3-L1 adipocytes were incubated with triiodothyronine at physiological dose (10nM) and supraphysiological doses (100nM or 1,000nM), or without triiodothyronine (control, C) for 0.5, 6, or 24 hours. Leptin and adiponectin mRNA was detected using real-time polymerase chain reaction (RT-PCR). One-way analyses of variance, Tukey’s test or Student’s t test, were used to analyze data, and significance level was set at 5%. Results Leptin levels decreased in the 1,000nM-dose group after 0.5 hour. Adiponectin levels dropped in the 10nM-dose group, but increased at the 100nM dose. After 6 hours, both genes were suppressed in all hormone concentrations. After 24 hours, leptin levels increased at 10, 100 and 1,000nM groups as compared to the control group; and adiponectin levels increased only in the 100nM group as compared to the control group. Conclusion These results demonstrated fast actions of triiodothyronine on the leptin and adiponectin expression, starting at 0.5 hour, at a dose of 1,000nM for leptin and 100nM for adiponectin. Triiodothyronine stimulated or inhibited the expression of adipokines in adipocytes at different times and doses which may be useful to assist in the treatment of obesity, assuming that leptin is increased and adiponectin is decreased, in obesity cases. PMID:25993072

  9. PPARgamma induces the insulin-dependent glucose transporter GLUT4 in the absence of C/EBPalpha during the conversion of 3T3 fibroblasts into adipocytes.

    PubMed Central

    Wu, Z; Xie, Y; Morrison, R F; Bucher, N L; Farmer, S R

    1998-01-01

    To define the molecular mechanisms that control GLUT4 expression during adipogenesis, NIH-3T3 fibroblasts ectopically expressing different adipogenic transcription factors (C/EBPbeta, C/EBPdelta, C/EBPalpha, and PPARgamma) under the control of a tetracycline-responsive inducible (C/EBPs) or a constitutive retroviral (PPARgamma) expression system were used. Enhanced production of C/EBPbeta (beta2 cell line), C/EBPbeta together with C/EBPdelta (beta/delta39 cell line), C/EBPalpha (alpha1 cell line), or PPARgamma (Pgamma2 cell line) in cells exposed to dexamethasone and the PPARgamma ligand ciglitazone (a thiazolidinedione) resulted in expression of GLUT4 mRNA as well as other members of the adipogenic gene program, including aP2 and adipsin. Focusing our studies on the beta/delta39 cells, we have demonstrated that C/EBPbeta along with C/EBPdelta in the presence of dexamethasone induces PPARgamma, adipsin, and aP2 mRNA production; however, GLUT4 mRNA is only expressed in cells exposed to ciglitazone. In addition, enhanced expression of a ligand-activated form of PPARgamma in the beta/delta39 fibroblasts stimulates synthesis of GLUT4 protein and gives rise to a population of adipocytic cells that take up glucose in direct response to insulin. C/EBPalpha is not expressed in the beta/delta39 cells under conditions that stimulate the adipogenic program. This observation suggests that PPARgamma alone or in combination with C/EBPbeta and C/EBPdelta is capable of activating GLUT4 gene expression. PMID:9421462

  10. Berberine reduces the expression of adipogenic enzymes and inflammatory molecules of 3T3-L1 adipocyte.

    PubMed

    Choi, Bong-Hyuk; Ahn, In-Sook; Kim, Yu-Hee; Park, Ji-Won; Lee, So-Young; Hyun, Chang-Kee; Do, Myoung-Sool

    2006-12-31

    Berberine (BBR), an isoquinoline alkaloid, has a wide range of pharmacological effects, yet its exact mechanism is unknown. In order to understand the anti-adipogenic effect of BBR, we studied the change of expression of several adipogenic enzymes of 3T3-L1 cells by BBR treatment. First, we measured the change of leptin and glycerol in the medium of 3T3-L1 cells treated with 1 micrometer, 5 micrometer and 10 micrometer concentrations of BBR. We also measured the changes of adipogenic and lipolytic factors of 3T3-L1. In 3T3-L1 cells, both leptin and adipogenic factors (SREBP-1c, C/EBP-alpha, PPAR-gamma, fatty acid synthase, acetyl-CoA carboxylase, acyl-CoA synthase and lipoprotein lipase) were reduced by BBR treatment. Glycerol secretion was increased, whereas expression of lipolytic enzymes (hormone-sensitive lipase and perilipin) mRNA was slightly decreased. Next, we measured the change of inflammation markers of 3T3-L1 cells by BBR treatment. This resulted in the down-regulation of mRNA level of inflammation markers such as TNF-alpha, IL-6, C- reactive protein and haptoglobin. Taken together, our data shows that BBR has both anti-adipogenic and anti-inflammatory effects on 3T3-L1 adipocytes, and the anti-adipogenic effect seems to be due to the down-regulation of adipogenic enzymes and transcription factors.

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

    PubMed

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

    2012-03-16

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

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

    SciTech Connect

    Takahashi, Nobuhiko; 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.

  13. 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.

  14. 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.

  15. Response of MC3T3-E1 osteoblasts, L929 fibroblasts, and J774 macrophages to fluoride surface-modified AZ31 magnesium alloy.

    PubMed

    Lozano, Rosa María; Pérez-Maceda, Blanca Teresa; Carboneras, Mónica; Onofre-Bustamante, Edgar; García-Alonso, María Cristina; Escudero, María Lorenza

    2013-10-01

    The present work evaluates the biocompatibility of a fluoride surface-modified AZ31 magnesium alloy (AZ31HF) with different cell lines that coexist in the implant environment to test its potential use as a biodegradable and absorbable biomaterial for bone repair. A clear stimulation of cell proliferation and an enhancement of the mitochondrial respiratory activity were observed when mouse osteoblasts (MC3T3-E1), fibroblasts (L929), and macrophages (J774) cell lines were cultured with the modified alloy. No significant change in apoptosis or viability rates was observed when osteoblasts and fibroblasts cultures were grown in the presence of this alloy. A proteomic analysis of the MC3T3-E1 cell extracts cultured in the presence of AZ31HF showed an overexpression of proteins related with the mineralization process, which is a necessary step for bone repair. An increase in the lactate dehydrogenase activity was observed in the MC3T3-E1 and J774 cell cultures that could be a response of the oxidative stress produced by the presence of the material. This stress could be related to the increase observed in the respiratory mitochondrial activity or respiratory burst measured in theses cultures that indicate damage in the cell membranes and subsequently some cell death. Results reported here, for and against AZ31HF, should be taken into account when considering the potential use of this modified alloy in bone repair applications.

  16. A quantitative description of the extension and retraction of surface protrusions in spreading 3T3 mouse fibroblasts.

    PubMed

    Albrecht-Buehler, G; Lancaster, R M

    1976-11-01

    We suggest a method of quantitating the motile actions of surface protrusions in spreading animal cells in culture. Its basis is the determination of the percentage of freshly plated cells which produce particle-free areas around them on a gold particle-coated glass cover slip within 50 min. Studying 3T3 cells with this assay, we found that the presence of Na+, K+, Cl-, and Mg++ or Ca++ in a neutral or slightly alkaline phosphate or bicarbonate buffered solution is sufficient to support the optimal particle removal by the cells for at least 50 min. Two metabolic inhibitors, 2,4-dinitrophenol and Na-azide, inhibit the particle removal. If D-glucose is added along with the inhibitors, particle removal can be restored, whereas the addition of three glucose analogues which are generally believed to be nonmetabolizable cannot restore the activity. Serum is not required for the mechanism(s) of the motile actions of surface protrusions in spreading 3T3 cells. However, it contains components which can neutralize the inhibitory actions of bovine serum albumin and several amino acids, particularly L-cystine or L-cystein and L-methionine. Furthermore, serum codetermines which of the major surface extension, filopodia, lamellipodia, or lobopodia, is predominantly active. We found three distinct classes of extracellular conditions under which the active surface projections are predominantly either lamellipodia, (sheetlike projections), lobopodia (blebs), or filopodia (microspikes). The quantitated dependencies on temperature, pH and the inhibition by cytochalasin B or the particle removal are very similar in all three cases. Preventing the cells from anchoring themselves for 15-20 min before plating in serum-free medium seems to stimulate particle removal threefold.

  17. 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.

  18. 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.

  19. Met promotes the formation of double minute chromosomes induced by Sei-1 in NIH-3T3 murine fibroblasts

    PubMed Central

    You, Jia; Wu, Di; Yu, Yang; Liu, Chang; Wang, Lei; Wang, Fei; Xu, Lu; Wang, Liqun; Wang, Nan; Tian, Xing; Wang, Falin; Liang, Hongbin; Gao, Yating; Cui, Xiaobo; Ji, Guohua; Bai, Jing; Yu, Jingcui; Meng, Xiangning; Jin, Yan; Sun, Wenjing; Guan, Xin-yuan; Zhang, Chunyu; Fu, Songbin

    2016-01-01

    Background Sei-1 is an oncogene capable of inducing double minute chromosomes (DMs) formation. DMs are hallmarks of amplification and contribute to oncogenesis. However, the mechanism of Sei-1 inducing DMs formation remains unelucidated. Results DMs formation significantly increased during serial passage in vivo and gradually decreased following culture in vitro. micro nuclei (MN) was found to be responsible for the reduction. Of the DMs-carrying genes, Met was found to be markedly amplified, overexpressed and highly correlated with DMs formation. Inhibition of Met signaling decreased the number of DMs and reduced the amplification of the DMs-carrying genes. We identified a 3.57Mb DMs representing the majority population, which consists of the 1.21 Mb AMP1 from locus 6qA2 and the 2.36 Mb AMP2 from locus 6qA2-3. Materials and Methods We employed NIH-3T3 cell line with Sei-1 overexpression to monitor and characterize DMs in vivo and in vitro. Array comparative genome hybridization (aCGH) and fluorescence in situ hybridization (FISH) were performed to reveal amplification regions and DMs-carrying genes. Metaphase spread was prepared to count the DMs. Western blot and Met inhibition rescue experiments were performed to examine for involvement of altered Met signaling in Sei-1 induced DMs. Genomic walking and PCR were adopted to reveal DMs structure. Conclusions Met is an important promotor of DMs formation. PMID:27494853

  20. Differential expression of fatty acid uptake in 3T3-L1 cells

    SciTech Connect

    Waggoner, D.; Bernlohr, D.A.

    1987-05-01

    Cultured 3T3-L1 cells have been used as a model system to investigate the mechanism of fatty acid uptake by adipose tissue. Using a 1:1 molar ratio of /sup 14/C-oleate and defatted bovine serum albumin (BSA), fatty acid (FA) uptake was quantitated at 4/sup 0/ and 37/sup 0/ as cell associated radioactivity. The profile of FA uptake in preadipocytes and adipocytes was biphasic; an initial rapid phase (1-20s) followed by a second slower phase (60-480s). At 37/sup 0/ the initial rate of FA accumulation in preadipocytes was identical to that in adipocytes, whereas the rate of accumulation during the second phase increased 7-fold (100 ..mu..M total FA) as a consequence of adipose conversion. When uptake measurements were made at 4/sup 0/ in adipocytes, the initial rate was identical to that at 37/sup 0/, however the rate of second phase decreased 5-fold. Incubation of /sup 14/C-BSA and nonradiolabeled FA with adipocyte monolayers (100 ..mu..M total FA) resulted in the rapid association (t/sub 1/2/ = 20s) of the BSA-FA complex with the cell surface. Incubation of 100, 10, and 1 ..mu..M total FA with adipocytes resulted in a 50-fold change in FA accumulation during the second phase. These results suggest that (1) FA uptake is significantly increased after differentiation, suggesting the participation of specialized proteins, (2) the temperature-insensitive initial FA accumulation can be attributed to rapid association of the BSA-FA complex to the cell surface, (3) the second phase of FA accumulation represents uptake.

  1. 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.

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

    SciTech Connect

    Hirai, Takao; Kaneshige, Kota; Kurosaki, Teruko; Nishio, Hiroaki

    2010-05-28

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

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

    PubMed

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

    2003-07-01

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

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

    PubMed

    Kim, Woo Kyoung; Kang, Nam E; Kim, Myung Hwan; Ha, Ae Wha

    2013-06-01

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

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

    PubMed Central

    1995-01-01

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

  6. Study of smart antibacterial PCL-xFe3 O4 thin films using mouse NIH-3T3 fibroblast cells in vitro.

    PubMed

    Pai B, Ganesh; Kulkarni, Ajay V; Jain, Shilpee

    2016-01-13

    Surface energy plays a major role in prokaryotic and eukaryotic cell interactions with biomedical devices. In the present study, poly(ε-caprolactone)-xFe3 O4 nanoparticles (PCL-xFO NPs; x = 0, 10, 20, 30, 40, 60 wt% FO concentration in PCL) composite thin films were developed for skin tissue regeneration. The surface properties in terms of roughness, surface energy, wettability of the thin films were altered with the incorporation of Fe3 O4 NPs. These thin films show antimicrobial properties and cyto-compatibility with NIH 3T3 mouse embryonic fibroblast cells. The porosity and thickness of the films were controlled by varying RPM of the spin coater. Interestingly, at 1000 RPM the roughness of the film decreased with increasing concentrations of FO NPs in PCL, whereas the surface energy increased with increasing FO NPs concentrations. Furthermore, the spreading of NIH-3T3 cells grown on PCL-xFO thin films was less as compared to control (TCPS), however cells overcame this effect after 48 h of seeding and cells spread similarly to those grown on TCPS after 48 h. Also, the incorporation of FO NPs in thin films induced inner membrane permeabilization in E. coli bacteria leading to bacterial cell death. The viability of E. coli bacteria decreased with increasing concentration of FO NPs in PCL. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2016.

  7. Exchange protein activated by cyclic AMP is involved in the regulation of adipogenic genes during 3T3-L1 fibroblasts differentiation.

    PubMed

    Gabrielli, Matías; Martini, Claudia N; Brandani, Javier N; Iustman, Laura J R; Romero, Damián G; del C Vila, María

    2014-02-01

    Adipogenesis is stimulated in 3T3-L1 fibroblasts by a combination of insulin, dexamethasone and isobutylmethylxanthine, IBMX, (I+D+M). Two transcription factors are important for the acquisition of the adipocyte phenotype, C/EBP beta (CCAT enhancer-binding protein beta) and PPAR gamma (peroxisome proliferator-activated receptor gamma). IBMX increases cAMP content, which can activate protein kinase A (PKA) and/or EPAC (exchange protein activated by cAMP). To investigate the importance of IBMX in the differentiation mixture, we first evaluated the effect of the addition of IBMX on the increase of C/EBP beta and PPAR gamma and found an enhancement of the amount of both proteins. IBMX addition (I+D+M) or its replacement with a cAMP analogue, dibutyryl-cAMP or 8-(4-chlorophenylthio)-2-O'-methyl-cAMP (8CPT-2-Me-cAMP), the latter activates EPAC and not PKA, remarkably increased PPAR gamma mRNA. However, neither I+D nor any of the inducers alone, increased PPAR gamma mRNA to a similar extent, suggesting the importance of the presence of both IBMX and I+D. It was also found that the addition of IBMX or 8CPT-2-Me-cAMP was able to increase the content of C/EBP beta with respect to I+D. In agreement with these findings, a microarray analysis showed that the presence of either 8CPT-2-Me-cAMP or IBMX in the differentiation mixture was able to upregulate PPAR gamma and PPAR gamma-activated genes as well as other genes involved in lipid metabolism. Our results prove the involvement of IBMX-cAMP-EPAC in the regulation of adipogenic genes during differentiation of 3T3-L1 fibroblasts and therfore contributes to elucidate the role of cyclic AMP in this process.

  8. 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

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

    PubMed Central

    2012-01-01

    Background Drugs of plant origin such as Arnica montana, Calendula officinalis or Hypericum perforatum have been frequently used to promote wound healing. While their effect on wound healing using preparations at pharmacological concentrations was supported by several in vitro and clinical studies, investigations of herbal homeopathic remedies on wound healing process are rare. The objective of this study was to investigate the effect of a commercial low potency homeopathic remedy Similasan® Arnica plus Spray on wound closure in a controlled, blind trial in vitro. Methods We investigated the effect of an ethanolic preparation composed of equal parts of Arnica montana 4x, Calendula officinalis 4x, Hypericum perforatum 4x and Symphytum officinale 6x (0712–2), its succussed hydroalcoholic solvent (0712–1) and unsuccussed solvent (0712–3) on NIH 3T3 fibroblasts. Cell viability was determined by WST-1 assay, cell growth using BrdU uptake, cell migration by chemotaxis assay and wound closure by CytoSelect ™Wound Healing Assay Kit which generated a defined “wound field”. All assays were performed in three independent controlled experiments. Results None of the three substances affected cell viability and none showed a stimulating effect on cell proliferation. Preparation (0712–2) exerted a stimulating effect on fibroblast migration (31.9%) vs 14.7% with succussed solvent (0712–1) at 1:100 dilutions (p < 0.001). Unsuccussed solvent (0712–3) had no influence on cell migration (6.3%; p > 0.05). Preparation (0712–2) at a dilution of 1:100 promoted in vitro wound closure by 59.5% and differed significantly (p < 0.001) from succussed solvent (0712–1), which caused 22.1% wound closure. Conclusion Results of this study showed that the low potency homeopathic remedy (0712–2) exerted in vitro wound closure potential in NIH 3T3 fibroblasts. This effect resulted from stimulation of fibroblasts motility rather than of their mitosis. PMID:22809174

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

    PubMed

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

    2012-07-01

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

  11. Effects of MicroRNA-23a on Differentiation and Gene Expression Profiles in 3T3-L1 Adipocytes

    PubMed Central

    Huang, Yong; Huang, Jinxiu; Qi, Renli; Wang, Qi; Wu, Yongjiang; Wang, Jing

    2016-01-01

    MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate growth, development, and programmed death of cells. A newly-published study has shown that miRNA-23a could regulate 3T3-L1 adipocyte differentiation. Here, we identified miRNA-23a as a negative regulator of 3T3-L1 adipocyte differentiation again. Over-expression of miRNA-23a inhibited differentiation and decreased lipogenesis as well as down-regulated mRNA and protein expression of both peroxisome proliferator-activated receptor (PPAR) γ and fatty acid binding protein (FABP) 4, whereas knock down of miRNA-23a showed the opposite effects on differentiation as well as increasing the number of apoptotic cells. Additionally, digital gene expression profiling sequencing (DGE-Seq) was used to assay changes in gene expression profiles following alterations in the level of miR-23a. In total, over-expression or knock down of miRNA-23a significantly changed the expression of 313 and 425 genes, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that these genes were mainly involved in the stress response, immune system, metabolism, cell cycle, among other pathways. Additionally, the signal transducer and activator of transcription 1 (Stat1) was shown to be a target of miRNA-23a by computational and dual-luciferase reporter assays that indicated Janus Kinase (Jak)-Stat signal pathway was implicated in regulating adipogenesis mediated by miRNA-23a in adipocytes. PMID:27783036

  12. Menaquinone-7 regulates the expressions of osteocalcin, OPG, RANKL and RANK in osteoblastic MC3T3E1 cells.

    PubMed

    Katsuyama, Hironobu; Otsuki, Takemi; Tomita, Masafumi; Fukunaga, Masao; Fukunaga, Tatsushige; Suzuki, Nobuo; Saijoh, Kiyofumi; Fushimi, Shigeko; Sunami, Shigeo

    2005-02-01

    Epidemiological studies show that dietary intake of natto, which contains significant amount of vitamin K(2), reduces the risk of bone formation loss. However, many confounding factors, such as calcium and isoflavone, are found in natto, because it is made from soybeans. In this study, the direct effects of MK-7, a vitamin K(2) analogue, were assessed in osteoblasts. Osteoblastic MC3T3E1 cells were cultured with or without MK-7 for 10 days and the number of cells was calculated. The cell count was not different between MK-7 treated cells and control cells for 1, 2, and 4 days. However, it was significantly suppressed in MK-7 treated cells at 10 days, suggesting that MK-7 suppressed cell proliferation. Real-time PCR analysis showed that mRNAs of osteocalcin (OC), osteoprotegerin (OPG), and the receptor activator of the NFkappaB ligand (RANKL) were induced after MK-7 administration to the culture medium. RANK mRNA expression was also enhanced by MK-7 administration. Immunocytochemical analysis showed that MK-7 increased the protein levels of OC and RANKL. RANK protein was also enhanced, but this induction was suppressed by anti-RANK antibody administration. This suppression was recovered when anti-RANK antibody and MK-7 were administered. These observations suggest that MK-7 may directly affect MC3T3E1 cells and stimulate osteoblastic differentiation, not proliferation.

  13. 3T3 fibroblasts induce cloned interleukin 3-dependent mouse mast cells to resemble connective tissue mast cells in granular constituency

    SciTech Connect

    Dayton, E.T.; Pharr, P.; Ogawa, M.; Serafin, W.E.; Austen, K.F.; Levi-Schaffer, F.; Stevens, R.L.

    1988-01-01

    As assessed by ultrastructure, histochemical staining, and T-cell dependency, in vitro-differentiated interleukin 3-dependent mouse mast cells are comparable to the mast cells that reside in the gastrointestinal mucosa but not in the skin or the serosal cavity of the mouse. The authors now demonstrate that when cloned interleukin 3-dependent mast cells are cocultured with mouse skin-derived 3T3 fibroblasts in the presence of WEHI-3 conditioned medium for 28 days, the mast cells acquire the ability to stain with safranin, increase their histamine content approx. 50-fold and their carboxypeptidase. A content approx. 100-fold, and augment approx. their biosynthesis of proteoglycans bearing /sup 35/S-labeled haparin relative to /sup 35/S-labeled chondroitin sulfate glycosaminoglycans. Thus, fibroblasts induce interleukin 3-dependent mouse mast cells to change phenotype from mucosal-like to connective tissue-like, indicating that the biochemical and functional characteristics of this mast cell type are strongly influenced by the connective tissue microenvironment.

  14. The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts.

    PubMed Central

    Kozma, R; Ahmed, S; Best, A; Lim, L

    1995-01-01

    The Ras-related protein Cdc42 plays a role in yeast cell budding and polarity. Two related proteins, Rac1 and RhoA, promote formation in mammalian cells of membrane ruffles and stress fibers, respectively, which contain actin microfilaments. We now show that microinjection of the related human Cdc42Hs into Swiss 3T3 fibroblasts induced the formation of peripheral actin microspikes, determined by staining with phalloidin. A proportion of these microspikes was found to be components of filopodia, as analyzed by time-lapse phase-contrast microscopy. The formation of filopodia was also found to be promoted by Cdc42Hs microinjection. This was followed by activation of Rac1-mediated membrane ruffling. Treatment with bradykinin also promoted formation of microspikes and filopodia as well as subsequent effects similar to that seen upon Cdc42Hs microinjection. These effects of bradykinin were specifically inhibited by prior microinjection of dominant negative Cdc42HsT17N, suggesting that bradykinin acts by activating cellular Cdc42Hs. Since filopodia have been ascribed an important sensory function in fibroblasts and are required for guidance of neuronal growth cones, these results indicate that Cdc42Hs plays an important role in determining mammalian cell morphology. PMID:7891688

  15. A comparative evaluation of photo-toxic effect of fractionated melanin and chlorpromazine hydrochloride on human (dermal fibroblasts and epidermal keratinocytes) and mouse cell line/s (fibroblast Balb/c 3T3).

    PubMed

    Rai, V; Dayan, N; Michniak-Kohn, B

    2011-03-01

    Fractionated melanin (Mel-HEV), a bleached version of natural melanin, offers protection against the high energy visible (HEV/UVA) and ultraviolet (specifically UVA) irradiation making it a potential compound to be added to skin care and sunscreen formulations and other cosmetic and personal care products. Chlorpromazine (CPZ) has been shown to exhibit photosensitivity and phototoxicity reaction in vitro and in vivo. Comparative evaluation of chemotoxicity and phototoxicity using Mel-HEV and CPZ (as positive control) was performed on mouse fibroblast cell line 'Balb/c 3T3'. This is the recommended method for evaluating the phototoxic potential of compounds under the European Center of Validation of Alternative Methods (ECVAM) guidelines (OECD, 2004). This study was expanded from a mouse cell line - Balb 3T3/c to two human cell lines - HDF and HEKn for two reasons: to compare the difference between the sensitivity and behavior of two fibroblast cell lines (Balb/c 3T3 vs. HDF) and to compare the differences between two fibroblast cell lines with the keratinocyte cell line (HDF & Balb/c 3T3 vs. HEKn). It was found that Balb/c 3T3 and HEKn were both sensitive to the phototoxic potential of CPZ. However, HDF showed insensitivity to phototoxic evaluation. The test compound, Mel-HEV, was found to be non-phototoxic. The mean toxic concentration (MTC) for CPZ during HEV and UVA exposure conditions was found to be similar using Balb/c 3T3 (36.25 μg/ml) and HEKn (39.99 μg/ml) showing that cells exhibit similar responses at HEV/UVA- conditions. However, Balb/c 3T3 showed more sensitivity to CPZ at HEV/UVA+ condition (MTC=0.87 μg/ml; mean PIF=55.33; MPE=0.395) than HEKn (MTC=5.35 μg/ml; PIF=7.61; MPE=0.276) making it the preferred cell line for phototoxicity evaluations.

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

    PubMed Central

    2013-01-01

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

  17. Regulation of Adipogenesis and Key Adipogenic Gene Expression by 1, 25-Dihydroxyvitamin D in 3T3-L1 Cells.

    PubMed

    Ji, Shuhan; Doumit, Matthew E; Hill, Rodney A

    2015-01-01

    The functions of 1, 25-dihydroxyvitamin D (1, 25-(OH)2D3) in regulating adipogenesis, adipocyte differentiation and key adipogenic gene expression were studied in 3T3-L1 preadipocytes. Five concentrations (0.01, 0.1, 1, 10, 100 nM) of 1, 25-(OH)2D3 were studied and lipid accumulation measured by Oil Red O staining and expression of adipogenic genes quantified using quantitative real-time PCR. Adipogenic responses to 1, 25-(OH)2D3 were determined on 6, and 12 h, and days 1-10 after induction of adipogenesis by a hormonal cocktail with or without 1, 25-(OH)2D3. In response to 1, 25-(OH)2D3 (1, 10, and 100 nM), lipid accumulation and the expression of PPARγ, C/EBPα, FABP4 and SCD-1 were inhibited through day 10, and vitamin D receptor expression was inhibited in the early time points. The greatest inhibitory effect was upon expression of FABP4. Expression of SREBP-1c was only affected on day 2. The lowest concentrations of 1, 25-(OH)2D3 tested did not affect adipocyte differentiation or adipogenic gene expression. The C/EBPα promoter activity response to 1, 25-(OH)2D3 was also tested, with no effect detected. These results indicate that 1, 25-(OH)2D3 inhibited adipogenesis via suppressing adipogenic-specific genes, and is invoked either during PPARγ activation or immediately up-stream thereof. Gene expression down-stream of PPARγ especially FABP4 was strongly inhibited, and we suggest that the role of 1, 25-(OH)2D3 in regulating adipogenesis will be informed by further studies of adipogenic-specific gene promoter activity.

  18. 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 μM 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.

  19. Regulation of glucose transport by insulin, bombesin, and bradykinin in Swiss 3T3 fibroblasts: Involvement of protein kinase C-dependent and -independent mechanisms

    SciTech Connect

    Dettori, C.; Meldolesi, J. )

    1989-05-01

    Glucose transport stimulation by insulin, bombesin, and bradykinin in Swiss 3T3 fibroblasts was compared with the phosphoinositide hydrolysis effects of the same stimulants in a variety of experimental paradigms known to affect generation and/or functioning of intracellular second messengers: short- and long-term treatments with phorbol dibutyrate, that cause activation and down-regulation of protein kinase C, respectively; cell loading with high (quin2), that causes clamping of (Ca{sup 2+}){sub i} near the resting level; poisoning with pertussis toxin, that affects the GTP binding proteins of the Go/Gi class; treatment with Ca{sup 2+} ionophores. ({sup 14}C) glucose transport stimulation by maximal (insulin) was affected by neither pertussis toxin nor protein kinase C down-regulation. This result correlates with the lack of effect of insulin on phosphoinositide hydrolysis. In contrast, part of the glucose transport responses induced by bombesin and bradykinin appeared to be mediated by protein kinase C in proportion with the stimulation induced by these peptides on the phosphoinositide hydrolysis. The protein kinase C-independent portion of the response to bradykinin was found to be inhibitable by pertussis toxin. This latter result might suggest an interaction between the bradykinin receptor and a glucose transporter, mediated by a protein of the Go/Gi class.

  20. Interactions between sub-10-nm iron and cerium oxide nanoparticles and 3T3 fibroblasts: the role of the coating and aggregation state

    NASA Astrophysics Data System (ADS)

    Safi, M.; Sarrouj, H.; Sandre, O.; Mignet, N.; Berret, J.-F.

    2010-04-01

    Recent nanotoxicity studies revealed that the physico-chemical characteristics of engineered nanomaterials play an important role in the interactions with living cells. Here, we report on the toxicity and uptake of cerium and iron oxide sub-10-nm nanoparticles by NIH/3T3 mouse fibroblasts. Coating strategies include low-molecular weight ligands (citric acid) and polymers (poly(acrylic acid), MW = 2000 g mol - 1). Electrostatically adsorbed on the surfaces, the organic moieties provide a negatively charged coating in physiological conditions. We find that most particles were biocompatible, as exposed cells remained 100% viable relative to controls. Only the bare and the citrate-coated nanoceria exhibit a slight decrease in mitochondrial activity at very high cerium concentrations (>1 g l - 1). We also observe that the citrate-coated particles are internalized/adsorbed by the cells in large amounts, typically 250 pg/cell after 24 h incubation for iron oxide. In contrast, the polymer-coated particles are taken up at much lower rates (<30 pg/cell). The strong uptake shown by the citrated particles is related to the destabilization of the dispersions in the cell culture medium and their sedimentation down to the cell membranes. In conclusion, we show that the uptake of nanomaterials by living cells depends on the coating of the particles and on its ability to preserve the colloidal nature of the dispersions.

  1. Poly(2-hydroxyethyl methacrylate)-b-poly(L-Lysine) cationic hybrid materials for non-viral gene delivery in NIH 3T3 mouse embryonic fibroblasts.

    PubMed

    Johnson, Renjith P; Uthaman, Saji; John, Johnson V; Heo, Min Seon; Park, In Kyu; Suh, Hongsuk; Kim, Il

    2014-09-01

    In order to develop efficient and nontoxic gene delivery vectors, a series of biocompatible block copolymers, poly[(2-hydroxyethyl methacrylate)40 -block-(L-lysine)n ] (n = 40, 80, 120, 150), are prepared by combining an atom transfer radical polymerization of 2-hydroxyethyl methacrylate with a ring-opening polymerization of N(ϵ) -(carbobenzoxy)-L-lysine N-carboxyanhydride. The block copolymers are successfully condensed with plasmid DNA (pDNA) into nanosized (<200 nm) polyplexes. As a representative sample, p(HEMA)40 -b-p(lys)150 is utilized to confirm the effective cellular and nuclear uptake of pDNA. The polymer/pDNA polyplexes exhibit very low cytotoxicity and enhanced transfection activity by being easily taken up into mouse embryonic fibroblast cell line (NIH 3T3). Thus, the chimeric block copolymers provide a means for developing versatile nonviral gene vectors harboring the ideal requirements of low cytotoxicity, good stability, and high transfection efficiency for gene therapy.

  2. Pharmaco-Phylogenetic Investigation of Methyl Gallate Isolated from Acacia nilotica (L.) Delile and Its Cytotoxic Effect on NIH3T3 Mouse Fibroblast.

    PubMed

    Mishra, Rohit K; Ramakrishna, M; Mishra, Vani; Pathak, Ashutosh; Rajesh, S; Sharma, Shivesh; Pandey, Avinash C; Nageswara Rao, G; Dikshit, Anupam

    2016-01-01

    Present exploration deals with the therapeutic perspective of methyl gallate isolated from the leaf extract of Acacia nilotica (L.) Delile in contrast to food-borne bacterial pathogen's viz., Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, Pseudomonas aeruginosa and Staphylococcus aureus with their evolutionary succession. The extract was subjected to phytochemical analysis and isolated compound was identified as methyl gallate using UV-vis, IR and NMR spectra. It was found most potent against K. pneumoniae with its minimum inhibition concentration (MIC) of 0.32 mg/ml and minimum bactericidal concentration (MBC) at 0.62 mg/ml. The correlation of MIC values with an evolutionary succession assists the relationship between their genetic and toxic properties. The cytotoxic pursuit of methyl gallate was additionally assessed over NIH3T3 mouse fibroblast by Neutral red (NR) uptake, MTT cell proliferation assay and did not disclose any relevant influence on cell viability as well as cell proliferation. As such, the methyl gallate extracted from the leaf of A. nilotica holds massive antibacterial aptitude and hands out towards a new paradigm for food and pharmaceutical industries.

  3. 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.

  4. Induction of fibrillin-2 and periostin expression in Osterix-knockdown MC3T3-E1 cells.

    PubMed

    Lee, So-Jeong; Lee, Eun-Hye; Park, Seung-Yoon; Kim, Jung-Eun

    2017-01-05

    Osteoporosis is the most common age-related bone disease that is characterized by an imbalance between osteoblasts for bone formation and osteoclasts for bone resorption. Anti-catabolic drugs have been developed to inhibit osteoclast activity and to prevent bone loss in osteoporosis. However, because it is difficult to increase bone mass in osteoporotic bone, it would be beneficial to simultaneously enhance osteoblast function and thus form bone. Osterix (Osx) is an essential transcription factor for osteoblast differentiation. To date, many studies have focused on discovering Osx target genes and on increasing osteoblast differentiation. However, Osx targets and the mechanisms controlling osteoblast differentiation, are not well known. Here, we generated stable Osx-knockdown cell lines by employing shRNA in MC3T3-E1 osteoblastic cells. Stable Osx-knockdown osteoblasts exhibited a significant reduction in cell differentiation and nodule formation, which was similar to the reduced osteoblast activity observed in an Osx-deficient mouse model. Using an Affymetrix GeneChip microarray, we determined the differential gene expression profile in response to Osx knockdown, which provided insight into molecular mechanisms underlying osteoblast differentiation. Of 2743 genes with roles in cell differentiation, 15 were upregulated and 2 were downregulated in Osx-knockdown osteoblasts. In particular, the expression of fibrillin-2 and periostin was significantly increased in Osx-knockdown osteoblasts compared to that in control cells, as validated by RT-PCR and quantitative real-time PCR. Finally, this study showed differential gene expression profiles for Osx-mediated osteoblast differentiation, suggesting that fibrillin-2 and periostin will be target candidates of Osx in osteoblast differentiation.

  5. Receptor-mediated inhibition of adenylate cyclase and stimulation of arachidonic acid release in 3T3 fibroblasts. Selective susceptibility to islet-activating protein, pertussis toxin

    SciTech Connect

    Murayama, T.; Ui, M.

    1985-06-25

    Thrombin exhibited diverse effects on mouse 3T3 fibroblasts. It (a) decreased cAMP in the cell suspension, (b) inhibited adenylate cyclase in the Lubrol-permeabilized cell suspension in a GTP-dependent manner, increased releases of (c) arachidonic acid and (d) inositol from the cell monolayer prelabeled with these labeled compounds, (e) increased /sup 45/Ca/sup 2 +/ uptake into the cell monolayer, and (f) increased /sup 86/Rb/sup +/ uptake into the cell monolayer in a ouabain-sensitive manner. Most of the effects were reproduced by bradykinin, platelet-activating factor, and angiotensin II. The receptors for these agonists are thus likely to be linked to three separate effector systems: the adenylate cyclase inhibition, the phosphoinositide breakdown leading to Ca/sup 2 +/ mobilization and phospholipase A2 activation, and the Na,K-ATPase activation. Among the effects of these agonists, (a), (b), (c), and (e) were abolished, but (d) and (f) were not, by prior treatment of the cells with islet-activating protein (IAP), pertussis toxin, which ADP-ribosylates the Mr = 41,000 protein, the alpha-subunit of the inhibitory guanine nucleotide regulatory protein (Ni), thereby abolishing receptor-mediated inhibition of adenylate cyclase. The effects (a), (c), (d), and (e) of thrombin, but not (b), were mimicked by A23187, a calcium ionophore. The effects of A23187, in contrast to those of receptor agonists, were not affected by the treatment of cells with IAP. Thus, the IAP substrate, the alpha-subunit of Ni, or the protein alike, may play an additional role in signal transduction arising from the Ca/sup 2 +/-mobilizing receptors, probably mediating process(es) distal to phosphoinositide breakdown and proximal to Ca/sup 2 +/ gating.

  6. Cytotoxicity and morphological transforming potential of cobalt nanoparticles, microparticles and ions in Balb/3T3 mouse fibroblasts: an in vitro model.

    PubMed

    Sabbioni, Enrico; Fortaner, Salvador; Farina, Massimo; Del Torchio, Riccardo; Olivato, Iolanda; Petrarca, Claudia; Bernardini, Giovanni; Mariani-Costantini, Renato; Perconti, Silvia; Di Giampaolo, Luca; Gornati, Rosalba; Di Gioacchino, Mario

    2014-06-01

    We previously described the behaviour of different cobalt forms, i.e., cobalt nanoparticles (CoNP), cobalt microparticles (CoMP) and cobalt ions (Co(2+)), in culture medium (dissolution, interaction with medium components, bioavailability) as well as their uptake and intracellular distribution in Balb/3T3 mouse fibroblasts (Sabbioni, Nanotoxicology, 2012). Here, we assess the cytotoxicity and morphological transformation of CoNP compared not only to Co(2+), but also to CoMP and to released Co products. Cytotoxicity reached maximum at 4-h exposure, with ranking CoMP > CoNP > Co(2+). However, if we consider toxicity as a function of intracellular Co, toxicity of the ionic forms seems to prevail over the particles. Co forms other than Co(2+) released from particles had toxicity intermediate between particles and ions. Alterations in concentrations of essential elements (Cu, Mg, Zn) in cells exposed to Co particles may contribute to toxicity. Both CoMP and CoNP (but not Co(2+) and other released Co forms) induced morphological transformation (CoMP > CoNP). This was dependent on reactive oxygen species production and lipid peroxidation, as indicated by inhibition of type III foci with ascorbic acid. The present results suggest that the previously demonstrated massive mitochondrial and nuclear Co internalisation and DNA adduct formation by CoMP and CoNP (Sabbioni, Nanotoxicology, 2012) induce toxicity and transformation. On the contrary, the role of ions released by particles in culture medium is negligible. Thus, both the chemical and the physical properties of Co particles contribute to cytotoxicity and morphological transformation.

  7. Dissociation of bradykinin-induced prostaglandin formation from phosphatidylinositol turnover in Swiss 3T3 fibroblasts: evidence for G protein regulation of phospholipase A/sub 2/

    SciTech Connect

    Burch, R.M.; Axelrod, J.

    1987-09-01

    In Swiss 3T3 fibroblasts bradykinin stimulated inositol phosphate (InsP) formation and prostaglandin E/sub 2/ (PGE/sub 2/) synthesis. The EC/sub 50/ values for stimulation of PGE/sub 2/ synthesis and InsP formation by bradykinin were similar, 200 pM and 275 pM, respectively. Guanosine-5'-(..gamma..-thio)triphosphate stimulated PGE/sub 2/ synthesis and InsP formation, and guanosine-5'-(..beta..-thio)diphosphate inhibited both PGE/sub 2/ synthesis and InsP formation stimulated by bradykinin. Neither bradykinin-stimulated PGE/sub 2/ synthesis nor InsP formation was sensitive to pertussis toxin. Phorbol ester, dexamethasone, and cycloheximide distinguished between bradykinin-stimulated PGE/sub 2/ synthesis and InsP formation. Phorbol 12-myristate 13-acetate enhanced bradykinin-stimulated PGE/sub 2/ synthesis but inhibited bradykinin-stimulated InsP formation. Pretreatment of cells with dexamethasone for 24 hr inhibited bradykinin-stimulated PGE/sub 2/ synthesis but was without effect on bradykinin-stimulated InsP formation. Cycloheximide inhibited on bradykinin-stimulated InsP formation. When bradykinin was added to cells prelabeled with (/sup 3/H) choline, the phospholipase A/sub 2/ products lysophosphatidylcholine and glycerophosphocholine were generated. The data suggest that bradykinin receptors are coupled by GTP-binding proteins to both phospholipase C and phospholipase A/sub 2/ and that phospholipase A/sub 2/ is the enzyme that catalyzes release of arachidonate for prostaglandin synthesis.

  8. Nickel-smelting fumes increased the expression of HIF-1α through PI3K/ERK pathway in NIH/3T3 cells

    PubMed Central

    Han, Dan; Yang, Yue; Zhang, Lin; Wang, Chao; Wang, Yue; Tan, Wen-Qiao; Hu, Xue-Ying; Wu, Yong-Hui

    2016-01-01

    Objective: The purpose of this study was to investigate the effects of Nickel (Ni) -smelting fumes on oncogenic proteins in vivo and in vitro. Methods: Ni fallout beside a Ni smelting furnace in a factory was sampled to study its toxic effect. The effects of Ni-smelting fumes on the regulation of PI3K and ERK signaling pathways and the important downstream hypoxia inducible factor, HIF-1α, were studied both in NIH/3T3 cells and in the lung tissue of rats. NIH/3T3 cell transformation induced by Ni-smelting fumes was also observed. Results: Ni-smelting fumes activated PI3K, p-AKT, p70S6K1, and ERK proteins and increased HIF-1α expression in a time- and dose-dependent manner. However, activation was suppressed when NIH/3T3 cells were pretreated with PI3K/AKT or ERK inhibitors. Ni-smelting fumes caused malignant transformation of NIH/3T3 cells. Conclusions: Ni-smelting fumes increased the expression of HIF-1α through the PI3K/ERK pathway in NIH/3T3 cells and induced malignant transformation in these cells indicating that Ni-smelting fumes may be a potential carcinogen in mammalian cells. PMID:27488040

  9. 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; Tao, Li; 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.

  10. C2C12 myotubes inhibit the proliferation and differentiation of 3T3-L1 preadipocytes by reducing the expression of glucocorticoid receptor gene.

    PubMed

    Chu, Weiwei; Wei, Wei; Yu, Shigang; Han, Haiyin; Shi, Xiaoli; Sun, Wenxing; Gao, Ying; Zhang, Lifan; Chen, Jie

    2016-03-25

    Obesity is a well-established risk factor to health for its relationship with insulin resistance, diabetes and metabolic syndrome. Myocyte-adipocyte crosstalk model plays a significant role in studying the interaction of muscle and adipose development. Previous related studies mainly focus on the effects of adipocytes on the myocytes activity, however, the influence of myotubes on the preadipocytes development remains unclear. The present study was carried out to settle this issue. Firstly, the co-culture experiment showed that the proliferation, cell cycle, and differentiation of 3T3-L1 preadipocytes were arrested, and the apoptosis was induced, by differentiated C2C12 myotubes. Next, the sensitivity of 3T3-L1 preadipocytes to glucocorticoids (GCs), which was well known as cell proliferation, differentiation, apoptosis factor, was decreased after co-cultured with C2C12 myotubes. What's more, our results showed that C2C12 myotubes suppressed the mRNA and protein expression of glucocorticoid receptor (GR) in 3T3-L1 preadipocytes, indicating the potential mechanism of GCs sensitivity reduction. Taken together, we conclude that C2C12 myotubes inhibited 3T3-L1 preadipocytes proliferation and differentiation by reducing the expression of GR. These data suggest that decreasing GR by administration of myokines may be a promising therapy for treating patients with obesity or diabetes.

  11. 3T3 cells in adipocytic conversion.

    PubMed

    O'Shea Alvarez, M S

    1991-01-01

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

  12. β₂ adrenergic receptor activation suppresses bone morphogenetic protein (BMP)-induced alkaline phosphatase expression in osteoblast-like MC3T3E1 cells.

    PubMed

    Yamada, Takayuki; Ezura, Yoichi; Hayata, Tadayoshi; Moriya, Shuichi; Shirakawa, Jumpei; Notomi, Takuya; Arayal, Smriti; Kawasaki, Makiri; Izu, Yayoi; Harada, Kiyoshi; Noda, Masaki

    2015-06-01

    β adrenergic stimulation suppresses bone formation in vivo while its actions in osteoblastic differentiation are still incompletely understood. We therefore examined the effects of β2 adrenergic stimulation on osteoblast-like MC3T3-E1 cells focusing on BMP-induced alkaline phosphatase expression. Morphologically, isoproterenol treatment suppresses BMP-induced increase in the numbers of alkaline phosphatase-positive small foci in the cultures of MC3T3-E1 cells. Biochemically, isoproterenol treatment suppresses BMP-induced enzymatic activity of alkaline phosphatase in a dose-dependent manner. Isoproterenol suppression of alkaline phosphatase activity is observed even when the cells are treated with high concentrations of BMP. With respect to cell density, isoproterenol treatment tends to suppress BMP-induced increase in alkaline phosphatase expression more in osteoblasts cultured at higher cell density. In terms of treatment protocol, continuous isoproterenol treatment is compared to cyclic treatment. Continuous isoproterenol treatment is more suppressive against BMP-induced increase in alkaline phosphatase expression than cyclic regimen. At molecular level, isoproterenol treatment suppresses BMP-induced enhancement of alkaline phosphatase mRNA expression. Regarding the mode of isoproterenol action, isoproterenol suppresses BMP-induced BRE-luciferase activity. These data indicate that isoproterenol regulates BMP-induced alkaline phosphatase expression in osteoblast-like MC3T3E1 cells.

  13. 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.

  14. Expression of progesterone receptor B is associated with G0/G1 arrest of the cell cycle and growth inhibition in NIH3T3 cells

    SciTech Connect

    Horiuchi, Shinji; Kato, Kiyoko . E-mail: kkatoh@tsurumi.beppu.kyushu-u.ac.jp; Suga, Shin; Takahashi, Akira; Ueoka, Yousuke; Arima, Takahiro; Nishida, Jun-ichi; Hachisuga, Toru; Kawarabayashi, Tatsuhiko; Wake, Norio

    2005-05-01

    Previously, we found a significant reduction of progesterone receptor B (PR-B) expression levels in the Ras-mediated NIH3T3 cell transformation, and re-expression of exogenous PR-B eliminated the tumorigenic potential. We hypothesized that this reduction is of biological significance in cell transformation. In the present study, we determined the correlation between PR-B expression and cell cycle progression. In synchronized NIH3T3 cells, we found an increase in PR-B protein and p27 CDK inhibitor levels in the G0/G1 phase and a reduction due to redistribution in the S and G2/M phases. The MEK inhibitor or cAMP stimulation arrested NIH3T3 cells in the G0/G1 phase of the cell cycle. The expression of PR-B and p27 CDK inhibitors was up-regulated by treatment with both the MEK inhibitor and cAMP. Treatment of synchronized cells with a PKA inhibitor in the presence of 1% calf serum resulted in a significant reduction in both PR-B and p27 levels. The decrease in the PR-B levels caused by anti-sense oligomers or siRNA corresponded to the reduction in p27 levels. PR-B overexpression by adenovirus infection induced p27 and suppressed cell growth. Finally, we showed that PR-B modulation involved in the regulation of NIH3T3 cell proliferation was independent of nuclear estrogen receptor (ER) activity but dependent on non-genomic ER activity.

  15. Bisphenol A increases aP2 expression in 3T3L1 by enhancing the transcriptional activity of nuclear receptors at the promoter

    PubMed Central

    Atlas, Ella; Pope, Louise; Wade, Mike G; Kawata, Alice; Boudreau, Adele; Boucher, Jonathan G

    2014-01-01

    Environmental pollutants, such as bisphenol A (BPA), have the potential to affect the differentiation processes and the biology of the adipose tissue. The 3T3-L1 model is one of the murine cell models used extensively for the investigation of the molecular events that govern the differentiation of adipocytes from a committed preadipocyte to a mature, lipid laden adipocyte. Most of the studies investigating the effects of BPA on preadipocyte differentiation have investigated the effects of this chemical in the presence of an optimal differentiation cocktail containing high concentrations of the synthetic glucocorticoid dexamethasone, conditions that result in 90% to 100% of differentiated adipocytes. Our studies employed the 3T3-L1 cell model in the absence of exogenous glucocorticoids. We show that BPA is able to increase the differentiation of the 3T3-L1 cells under these conditions. Furthermore, the effect of BPA was observed in the absence of the synthetic glucocorticoid (dexamethasone), a hormone known to be required for the differentiation of the 3T3-L1 cells. In addition, BPA upregulated the mRNA expression and protein levels of the terminal marker of adipogenesis the fatty acid binding protein (aP2) in these cells. Interestingly, the known modulators of adipogenesis such as the peroxisome proliferator-activated receptor (PPAR) γ or CCAAT enhancer binding protein (C/EBP) α were not elevated at the mRNA or protein level in response to BPA. Furthermore, BPA upregulated the expression levels of the marker of adipogenesis aP2, through an effect on the transcriptional activity of C/EBPδ and the glucocorticoid receptor (GR) at its promoter. PMID:25068083

  16. 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.

  17. 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.

  18. Genotoxicity and morphological transformation induced by cobalt nanoparticles and cobalt chloride: an in vitro study in Balb/3T3 mouse fibroblasts.

    PubMed

    Ponti, Jessica; Sabbioni, Enrico; Munaro, Barbara; Broggi, Francesca; Marmorato, Patrick; Franchini, Fabio; Colognato, Renato; Rossi, François

    2009-09-01

    Nanotechnology is an emerging field that involves the development, manufacture and measurement of materials and systems in the submicron to nanometer range. Its development is expected to have a large socio-economical impact in practically all fields of industrial activity. However, there is still a lack of information about the potential risks of manufactured nanoparticles for the environment and for human health. In this work, we studied the cytotoxicity, genotoxicity and morphological transforming activity of cobalt nanoparticles (Co-nano) and cobalt ions (Co(2+)) in Balb/3T3 cells. We also evaluated Co-nano dissolution in culture medium and cellular uptake of both Co-nano and Co(2+). Our results indicated dose-dependent cytotoxicity, assessed by colony-forming efficiency test, for both compounds. The toxicity was higher for Co-nano than for Co(2) after 2 and 24 h of exposure, while dose-effect relationships were overlapping after 72 h. Statistically significant results were observed for Co-nano with the micronucleus test and the comet assay, while for Co(2+) positive results were observed only with the latter. In addition, even when Co-nano was genotoxic (at >1 microM), no evident dose-dependent effect was observed. Concerning morphological transformation, we found a statistically significant increase in the formation of type III foci (morphologically transformed colonies) only for Co-nano. Furthermore, we observed a higher cellular uptake of Co-nano compared with Co(2+).

  19. Involvement of JNK/NFκB Signaling Pathways in the Lipopolysaccharide-Induced Modulation of Aquaglyceroporin Expression in 3T3-L1 Cells Differentiated into Adipocytes

    PubMed Central

    Chiadak, Jeanne Durendale; Arsenijevic, Tatjana; Gregoire, Francoise; Bolaky, Nargis; Delforge, Valerie; Perret, Jason; Delporte, Christine

    2016-01-01

    Aquaglyceroporins, belonging to the family of aquaporins (AQPs), are integral plasma membrane proteins permeable to water and glycerol that have emerged as key players in obesity. The aim of this study was to investigate the expression profile of AQPs in undifferentiated and differentiated 3T3-L1 cells and to investigate the changes in expression of aquaglyceroporins in 3T3-L1 cells differentiated into adipocytes and subjected to lipopolysaccharide (LPS) mimicking inflammation occurring during obesity. Furthermore, the study aimed at identifying the signaling cascade involved in the regulation of aquaglyceroporins expression upon LPS stimulation. 3T3-L1 cells were grown as undifferentiated cells (UDC; preadipocytes) or cells differentiated into adipocytes (DC, adipocytes). DC were incubated in the presence or absence of LPS with or without inhibitors of various protein kinases. AQPs mRNA expression levels were measured by real-time quantitative polymerase chain reaction (RT-qPCR). AQP1, AQP2, AQP3, AQP9 and AQP11 mRNA were expressed in both UDC and DC, whereas AQP4, AQP7 and AQP8 mRNA were expressed only in DC. In DC, LPS up-regulated AQP3 mRNA levels (p < 0.05) compared to control; these effects were inhibited by CLI095, SP600125 and BAY11-7082 (p < 0.05). LPS decreased both AQP7 and AQP11 mRNA levels (p < 0.01) in DC as compared to control; this decrease was inhibited by CLI095 and BAY11-7082 (p < 0.05) and additionally by SP00125 for AQP7 (p < 0.05). SB203580 had no effect on LPS-induced AQP3, AQP7 and AQP11 mRNA levels modulations. In conclusion, our results clearly show that many AQPs are expressed in murine 3T3-L1 adipocytes. Moreover, in DCs, LPS led to decreased AQP7 and AQP11 mRNA levels but to increased AQP3 mRNA levels, resulting from the Toll-like receptor 4 (TLR4)-induced activation of JNK and/or NFκB pathway. PMID:27763558

  20. Acetate alters expression of genes involved in beige adipogenesis in 3T3-L1 cells and obese KK-Ay mice.

    PubMed

    Hanatani, Satoko; Motoshima, Hiroyuki; Takaki, Yuki; Kawasaki, Shuji; Igata, Motoyuki; Matsumura, Takeshi; Kondo, Tatsuya; Senokuchi, Takafumi; Ishii, Norio; Kawashima, Junji; Kukidome, Daisuke; Shimoda, Seiya; Nishikawa, Takeshi; Araki, Eiichi

    2016-11-01

    The induction of beige adipogenesis within white adipose tissue, known as "browning", has received attention as a novel potential anti-obesity strategy. The expression of some characteristic genes including PR domain containing 16 is induced during the browning process. Although acetate has been reported to suppress weight gain in both rodents and humans, its potential effects on beige adipogenesis in white adipose tissue have not been fully characterized. We examined the effects of acetate treatment on 3T3-L1 cells and in obese diabetic KK-Ay mice. The mRNA expression levels of genes involved in beige adipocyte differentiation and genes selectively expressed in beige adipocytes were significantly elevated in both 3T3-L1 cells incubated with 1.0 mM acetate and the visceral white adipose tissue from mice treated with 0.6% acetate for 16 weeks. In KK-Ay mice, acetate reduced the food efficiency ratio and increased the whole-body oxygen consumption rate. Additionally, reduction of adipocyte size and uncoupling protein 1-positive adipocytes and interstitial areas with multilocular adipocytes appeared in the visceral white adipose tissue of acetate-treated mice, suggesting that acetate induced initial changes of "browning". In conclusion, acetate alters the expression of genes involved in beige adipogenesis and might represent a potential therapeutic agent to combat obesity.

  1. Acetate alters expression of genes involved in beige adipogenesis in 3T3-L1 cells and obese KK-Ay mice

    PubMed Central

    Hanatani, Satoko; Motoshima, Hiroyuki; Takaki, Yuki; Kawasaki, Shuji; Igata, Motoyuki; Matsumura, Takeshi; Kondo, Tatsuya; Senokuchi, Takafumi; Ishii, Norio; Kawashima, Junji; Kukidome, Daisuke; Shimoda, Seiya; Nishikawa, Takeshi; Araki, Eiichi

    2016-01-01

    The induction of beige adipogenesis within white adipose tissue, known as “browning”, has received attention as a novel potential anti-obesity strategy. The expression of some characteristic genes including PR domain containing 16 is induced during the browning process. Although acetate has been reported to suppress weight gain in both rodents and humans, its potential effects on beige adipogenesis in white adipose tissue have not been fully characterized. We examined the effects of acetate treatment on 3T3-L1 cells and in obese diabetic KK-Ay mice. The mRNA expression levels of genes involved in beige adipocyte differentiation and genes selectively expressed in beige adipocytes were significantly elevated in both 3T3-L1 cells incubated with 1.0 mM acetate and the visceral white adipose tissue from mice treated with 0.6% acetate for 16 weeks. In KK-Ay mice, acetate reduced the food efficiency ratio and increased the whole-body oxygen consumption rate. Additionally, reduction of adipocyte size and uncoupling protein 1-positive adipocytes and interstitial areas with multilocular adipocytes appeared in the visceral white adipose tissue of acetate-treated mice, suggesting that acetate induced initial changes of “browning”. In conclusion, acetate alters the expression of genes involved in beige adipogenesis and might represent a potential therapeutic agent to combat obesity. PMID:27895388

  2. 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-α (sTNF-α), which is released from transmembranous TNF-α by TNF-α 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-α 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-α 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

  3. 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

  4. PDGF-induced receptor phosphorylation and phosphoinositide hydrolysis are unaffected by protein kinase C activation in mouse swiss 3T3 and human skin fibroblasts

    SciTech Connect

    Sturani, E.; Vicentini, L.M.; Zippel, R.; Toschi, L.; Pandiella-Alonso, A.; Comoglio, P.M.; Meldolesi, J.

    1986-05-29

    Short (1-10 min) pretreatment of intact cells with activators of protein kinase C (e.g. phorbol-12 myristate, 13-acetate, PMA) affects the activity of a variety of surface receptors (for growth factors, hormones and neurotransmitters), with inhibition of transmembrane signal generation. In two types of fibroblasts it is demonstrated that the PDGF receptor is unaffected by PMA. Exposure to PMA at concentrations up to 100 nM for 10 min failed to inhibit either one of the agonist-induced, receptor-coupled responses of PDGF: the autophosphorylation of receptor molecules at tyrosine residues, and the hydrolysis of membrane polyphosphoinositides. In contrast, the EGF receptor autophosphorylation (in A 431 cells) and the bombesin-induced phosphoinositide hydrolysis were readily inhibited by PMA.

  5. Red yeast rice extracts suppress adipogenesis by down-regulating adipogenic transcription factors and gene expression in 3T3-L1 cells.

    PubMed

    Jeon, Taeil; Hwang, Seong Gu; Hirai, Shizuka; Matsui, Tohru; Yano, Hideo; Kawada, Teruo; Lim, Beoung Ou; Park, Dong Ki

    2004-11-12

    The effects of red yeast rice extracts (RE) on adipocyte differentiation of 3T3-L1 cells were studied. RE were extracted from embryonic rice fermented with red yeast (Monascus ruber). These extracts significantly decreased glycerol-3-phosphate dehydrogenase (GPDH) activity and lipid accumulation, a marker of adipogenesis, in a dose-dependent manner. Moreover, mRNA expression levels of both CCAAT/enhancer-binding protein (C/EBP) alpha and peroxisome proliferator-activated receptor (PPAR) gamma, the key adipogenic transcription factors, were markedly decreased by RE. RE also inhibited the expression of PPARgamma at protein levels. RE decreased significantly gene expression of adipocyte fatty acid binding protein (aP2) and leptin, which are adipogenic marker proteins and C/EBPalpha and PPARgamma target genes. These results suggest that the inhibitory effect of RE on adipocyte differentiation might be mediated through the down-regulated expression of adipogenic transcription factors and other specific genes.

  6. 27-Hydroxycholesterol suppresses lipid accumulation by down-regulating lipogenic and adipogenic gene expression in 3T3-L1 cells.

    PubMed

    Shirouchi, Bungo; Kashima, Kentaro; Horiuchi, Yasutaka; Nakamura, Yuki; Fujimoto, Yumiko; Tong, Li-Tao; Sato, Masao

    2016-03-17

    Cholesterol oxidation products (oxycholesterols) are produced from cholesterol by automatic and/or enzymatic oxidation of the steroidal backbone and side-chain. Oxycholesterols are present in plasma and serum, suggesting that oxycholesterols are related to the development and progression of various diseases. However, limited information is available about the absolute amounts of oxycholesterols in organs and tissues, and the physiological significance of oxycholesterols in the body. In the present study, we quantified the levels of 13 oxycholesterols in white adipose tissue (WAT) of mice and then evaluated correlations between each oxycholesterol level and WAT weight. The sum of the levels of 13 oxycholesterols in WAT (white adipose tissue) was 15.9 ± 3.4 μg/g of WAT weight and approximately 1 % of cholesterol level. Among oxycholesterols, the levels of 27-hydroxycholesterol (27-OH), an endogenous oxycholesterol produced by enzymatic oxidation, and the relative WAT weights were significantly negatively correlated. Next, we evaluated the effects of 27-OH on lipogenesis and adipogenesis in 3T3-L1 cells. TO901317 (TO), a potent and selective agonist for LXRα, significantly increased intracellular TAG contents, while 27-OH significantly reduced the contents to half when compared with control (DMSO) and completely abolished the effect of TO. In addition, 27-OH significantly reduced the mRNA levels of lipogenic (LXRα and FAS) and adipogenic genes (PPARγ and aP2) during adipocyte maturation of 3T3-L1 cells. In conclusion, our results indicate that 27-OH suppresses lipid accumulation by down-regulating lipogenic and adipogenic gene expression in 3T3-L1 cells.

  7. Nonivamide enhances miRNA let-7d expression and decreases adipogenesis PPARγ expression in 3T3-L1 cells.

    PubMed

    Rohm, Barbara; Holik, Ann-Katrin; Kretschy, Nicole; Somoza, Mark M; Ley, Jakob P; Widder, Sabine; Krammer, Gerhard E; Marko, Doris; Somoza, Veronika

    2015-06-01

    Red pepper and its major pungent principle, capsaicin (CAP), have been shown to be effective anti-obesity agents by reducing energy intake, enhancing energy metabolism, decreasing serum triacylglycerol content, and inhibiting adipogenesis via activation of the transient receptor potential cation channel subfamily V member 1 (TRPV1). However, the binding of CAP to the TRPV1 receptor is also responsible for its pungent sensation, strongly limiting its dietary intake. Here, the effects of a less pungent structural CAP-analog, nonivamide, on adipogenesis and underlying mechanisms in 3T3-L1 cells were studied. Nonivamide was found to reduce mean lipid accumulation, a marker of adipogenesis, to a similar extent as CAP, up to 10.4% (P < 0.001). Blockage of the TRPV1 receptor with the specific inhibitor trans-tert-butylcyclohexanol revealed that the anti-adipogenic activity of nonivamide depends, as with CAP, on TRPV1 receptor activation. In addition, in cells treated with nonivamide during adipogenesis, protein levels of the pro-adipogenic transcription factor peroxisome-proliferator activated receptor γ (PPARγ) decreased. Results from miRNA microarrays and digital droplet PCR analysis demonstrated an increase in the expression of the miRNA mmu-let-7d-5p, which has been associated with decreased PPARγ levels.

  8. Effect of in vitro administered 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on DNA-binding activities of nuclear transcription factors in NIH-3T3 mouse fibroblasts.

    PubMed

    Hwang, Seong Gu; Sasagawa, Hiromi; Matsumura, Fumio

    2007-01-01

    TCDD is a very toxic environmental contaminant which is known to cause a variety of toxic symptoms in many species. Because of a myriad of biochemical changes TCDD is known to induce in many test animals, it has been difficult to pinpoint the causative event common to all those symptoms in different species. One of the research avenues we have been following is identification of the pattern of TCDD-induced changes in DNA binding characteristics of nuclear transcription factors (NTFs), each of which has the property to trigger a set of coordinated changes in many gene expressions. Since in our previous work we studied animals affected by TCDD in vivo using gel mobility shift assay approached with32P labeled oligonucleotide probes, we examined in the current study the possibility whether we could establish an equivalent in vitro system in NIH-3T3 mouse fibroblast cells so as to be able to learn the similarities and the dissimilarities of TCDD-induced responses of NTFs between in vitro and in vivo. The results indicated that, for a large part, this in vitro test system could reasonably reproduce the pattern of changes occurring in vivo at the early stages of TCDD's action in terms of induced changes in binding of thes NTSs to DNA. The key features were TCDD induced upregulation of NTFs binding to the response elements for AP-1, dioxin (DRE) and T3 (thyroid hormone) and down-regulation of those to response elements (REs) for c-Myc, Sp-1 and retinoic acid receptor (RARE). However, the time course required the changes in DNA binding activity was much shorter in vitro. To study the basic cause for such changes in NTF binding, we studied the effects of exogenously added EGF, forskolin, TPA (12-0-tetradecanoylphorbol acetate) and TNFalpha on the expression of TCDD's action on some of these NTFs. The results showed that these agents indeed greatly influence the outcome. The most influential agents were TNFalpha, forskolin and EGF. These results indicate that this in vitro

  9. PPARα agonist fenofibrate attenuates TNF-α-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-α (PPARα) 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 PPARα in inflammation is mediated by SIRT1 remains unclear. In this study, we aimed to determine the effect of PPARα 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-α (TNF-α)-stimulated adipocytes, and these effects of fenofibrate were reversed by PPARα antagonist GW6471. Moreover, SIRT1 inhibitors sirtinol/nicotinamide (NAM) or knockdown of SIRT1 could attenuate the effect of fenofibrate on TNF-α-induced CD40 expression in adipocytes. Importantly, NF-κB 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-κB p65 (Ac-NF-κB p65) in TNF-α-stimulated adipocytes, and the effect of fenofibrate was abolished by SIRT1 inhibition. In addition, fenofibrate up-regulated SIRT1 expression through AMPK in TNF-α-stimulated adipocytes. Taken together, these findings indicate that PPARα agonist fenofibrate inhibits TNF-α-induced CD40 expression in 3T3-L1 adipocytes via the SIRT1-dependent signaling pathway. -- Highlights: • Fenofibrate up-regulates SIRT1 expression in TNF-α-stimulated adipocytes. • Fenofibrate inhibits CD40 expression through SIRT1 in adipocytes. • The effects of fenofibrate on CD40 and SIRT1 expressions are dependent on PPARα. • Fenofibrate inhibits CD40 expression via SIRT1-dependent deacetylation of NF-κB. • Fenofibrate increases SIRT1 expression through PPARα and AMPK in adipocytes.

  10. 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%), β-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.

  11. Factors influencing the expression of endogenous reverse transcriptases and viral-like 30 elements in mouse NIH3T3 cells.

    PubMed

    Tzavaras, Theodore; Eftaxia, Sofia; Tavoulari, Sotiria; Hatzi, Paraskevi; Angelidis, Charalambos

    2003-10-01

    Retroviral reverse transcriptase (RT) plays a definite role in retroviral life cycle and is essential for the process of retrotransposition. We investigated the RNA expression of endogenous reverse transcriptases (enRTs) in the NIH3T3 mouse genome using, as a probe, a mixture of RT-PCR generated reverse transcriptase products potentially detecting a large number of RTs following treatment with different agents. We found that the expression of enRTs is induced approximately 500-fold following 5'-azacytidine-treatment. Amongst steroid hormones used such as estradiol, diethylstilbestrol, progesterone and dexamethasone only the latter was effective in inducing enRTs up to 4-fold at a concentration of 10(-7) M. Expression of a mouse dominant-negative form of p53 protein in cell clones resulted in induction of 20- to 50-fold, whereas C2-ceramide in a 4-fold induction at concentrations of 20-80 micro M. In a parallel analysis, the respective expression of the transposable viral-like 30 elements (VL30s) was also measured. Their expression was induced up to 50-fold by 5'-azacytidine, overexpression of the p53 gene and C2-ceramide at 80 micro M. It was also induced approximately 3- to 5-fold following estradiol, diethylstilbestrol or progesterone treatment and 30-fold by dexamethasone. Collectively, our results suggest that such stimuli inducing enRTs might play a role in the activation of transcription and retrotransposition of VL30.

  12. Loss of platelet-derived growth factor-stimulated phospholipase activity in NIH-3T3 cells expressing the EJ-ras oncogene

    SciTech Connect

    Benjamin, C.W.; Tarpley, W.G.; Gorman, R.R.

    1987-01-01

    Data indicating that the 21-kDa protein (p21) Harvey-ras gene product shares sequence homology with guanine nucleotide-binding proteins (G proteins) has stimulated research on the influence(s) of p21 on G-protein-regulated systems in vertebrate cells. Previous work demonstrated that NIH-3T3 mouse cells expressing high levels of the cellular ras oncogene isolated from the EJ human bladder carcinoma (EJ-ras) exhibited reduced hormone-stimulated adenylate cyclase activity. The authors now report that in these cells another enzyme system thought to be regulated by G proteins is inhibited, namely phospholipases A/sub 2/ and C. NIH-3T3 cells incubated in plasma-derived serum release significant levels of prostaglandin E/sub 2/ (PGE/sub 2/) as determined by radioimmunoassay when exposed to platelet-derived growth factor (PDGF) at 2 units/ml. The lack of PDGF-stimulated PGE/sub 2/ release from EJ-ras-transfected cells is not due to a defect in the prostaglandin cyclooxygenase enzyme, since incubation of control cells and EJ-ras-transfected cells in 0.33, 3.3, or 33 ..mu..M arachidonate resulted in identical levels of PGE/sub 2/ release. The lack of PDGF-stimulated PGE/sub 2/ release from EJ-ras-transfected cells also does not result from the loss of functional PDGF receptors. EJ-ras-transformed cells bind 70% as much /sup 125/I-labeled PDGF as control cells and are stimulated to incorporate (/sup 3/H)thymidine and to proliferate after exposure to PDGF. Determination of total water-soluble inositolphospholipids and changes in the specific activities of phosphatidylcholine in control and EJ-ras-transfected cells demonstrated that PDGF-stimulated phospholipase C and A/sub 2/ activities are inhibited in the EJ-ras-transfected cells.

  13. TNF-alpha inhibits 3T3-L1 adipocyte differentiation without downregulating the expression of C/EBPbeta and delta.

    PubMed

    Kurebayashi, S; Sumitani, S; Kasayama, S; Jetten, A M; Hirose, T

    2001-04-01

    Tumor necrosis factor-alpha (TNF-alpha) has been reported to inhibit adipocyte differentiation in which multiple transcription factors including CCAAT enhancer binding proteins (C/EBPs) and peroxisome proliferator-activated receptor (PPAR) gamma play an important role. Induction of C/EBPalpha and PPARgamma, which regulate the expression of many adipocyte-related genes, is dependent on the expression of C/EBPbeta and C/EBPdelta at the early phase of adipocyte differentiation. To elucidate the mechanism by which TNF-alpha inhibits adipocyte differentiation, we examined the effect of TNF-alpha on the expression of these transcription factors in mouse 3T3-L1 preadipocytes. TNF-alpha did not abrogate the induction of C/EBPbeta and C/EBPdelta in response to differentiation stimuli. In fully differentiated adipocytes, TNF-alpha rapidly induced C/EBPbeta and C/EBPdelta, whereas it downregulated the expression of C/EBPalpha and PPARgamma. Our results suggest that TNF-alpha inhibits adipocyte differentiation independently of the downregulation of C/EBPbeta and C/EBPdelta.

  14. Expression of cell adhesion and differentiation related genes in MC3T3 osteoblasts plated on titanium alloys: role of surface properties.

    PubMed

    Sista, Subhash; Wen, Cuie; Hodgson, Peter D; Pande, Gopal

    2013-04-01

    It is important to understand the cellular and molecular events that take place at the cell-material interface of implants used for bone repair. An understanding of the mechanisms involved in the initial stages of osteoblast interactions with the surface of the implant material is fundamental in deciding the fate of the cells that come in contact with it. In this study, we compared the relative gene expression of markers that are known to be associated with cell adhesion and differentiation in MC3T3 osteoblast cells, at various time points after plating the cells on surfaces of titanium (Ti) and its two alloys, titanium-zirconium (TiZr) and titanium-niobium (TiNb) by using Quantitative Real Time Polymerase Chain Reaction (RT-PCR). Our analysis indicated that expression of adhesion supporting genes was higher on TiZr surface as compared to Ti and TiNb. The behavior of these genes is possibly driven by a higher surface energy of TiZr. However no significant difference in the expression of differentiation related genes could be seen between the two alloys, although on both substrates it was higher as compared to unalloyed Ti. We propose that substrate composition of the alloys can influence the adhesion and differentiation related gene expression and that Ti alloys are better substrates for inducing osteogenesis as compared to unalloyed Ti.

  15. α-Mangostin Improves Glucose Uptake and Inhibits Adipocytes Differentiation in 3T3-L1 Cells via PPARγ, 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 α-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 PPARγ, GLUT4, and leptin expressions. α-Mangostin was found to inhibit cytoplasmic lipid accumulation and adipogenic differentiation. Cells treated with 50 μM of α-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 α-mangostin significantly improved the glucose uptake (P < 0.05) with highest activity found at 25 μM. In addition, α-mangostin increased the amount of free fatty acids (FFA) released. The highest glycerol release level was observed at 50 μM of α-mangostin. qRT-PCR analysis showed reduced lipid accumulation via inhibition of PPARγ gene expression. Induction of glucose uptake and free fatty acid release by α-mangostin were accompanied by increasing mRNA expression of GLUT4 and leptin. These evidences propose that α-mangostin might be possible candidate for the effective management of obesity in future. PMID:25873982

  16. 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

  17. C(2)-ceramide influences the expression and insulin-mediated regulation of cyclic nucleotide phosphodiesterase 3B and lipolysis in 3T3-L1 adipocytes.

    PubMed

    Mei, Jie; Holst, Lena Stenson; Landström, Tova Rahn; Holm, Cecilia; Brindley, David; Manganiello, Vincent; Degerman, Eva

    2002-03-01

    Cyclic nucleotide phosphodiesterase (PDE) 3B plays an important role in the antilipolytic action of insulin and, thereby, the release of fatty acids from adipocytes. Increased concentrations of circulating fatty acids as a result of elevated or unrestrained lipolysis cause insulin resistance. The lipolytic action of tumor necrosis factor (TNF)-alpha is thought to be one of the mechanisms by which TNF-alpha induces insulin resistance. Ceramide is the suggested second messenger of TNF-alpha action, and in this study, we used 3T3-L1 adipocytes to investigate the effects of C(2)-ceramide (a short-chain ceramide analog) on the expression and regulation of PDE3B and lipolysis. Incubation of adipocytes with 100 micromol/l C(2)-ceramide (N-acetyl-sphingosine) resulted in a time-dependent decrease of PDE3B activity, accompanied by decreased PDE3B protein expression. C(2)-ceramide, in a time- and dose-dependent manner, stimulated lipolysis, an effect that was blocked by H-89, an inhibitor of protein kinase A. These ceramide effects were prevented by 20 micromol/l troglitazone, an antidiabetic drug. In addition to downregulation of PDE3B, the antilipolytic action of insulin was decreased by ceramide treatment. These results, together with data from other studies on PDE3B and lipolysis in diabetic humans and animals, suggest a novel pathway by which ceramide induces insulin resistance. Furthermore, PDE3B is demonstrated to be a target for troglitazone action in adipocytes.

  18. Inhibitory effects of harpagoside on TNF-α-induced pro-inflammatory adipokine expression through PPAR-γ 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)-α-induced inflammatory adipokine expression and its underlying signaling pathways in differentiated 3T3-L1 cells. Harpagoside significantly inhibited TNF-α-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)-γ. 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.

  19. Traditional medicine yanggyuksanhwa-tang inhibits adipogenesis and suppresses proliferator-activated receptor-gamma expression in 3T3-L1 cells

    PubMed Central

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

    2015-01-01

    Background: Yanggyuksanhwa-tang (YGSHT) is a specific traditional Korean herbal formula for Soyangin according to Sasang constitutional philosophy. Although its biological activities against inflammation and cerebral infarction have been reporting, there is no information about the adipogenic activity of YGSHT. In the present study, we investigated the anti-adipogenic activity of YGSHT to evaluate effects of YGSHT on adipogenesis in vitro. Materials and Methods: Using 3T3-L1 preadipocytes, we induced the cellular differentiation into adipocytes by adding insulin. Anti-adipogenic activity of YGSHT was measured by oil red O staining, triglyceride assay, glycerol-3-phosphate dehydrogenase (GPDH) activity test, and leptin assay. Results: YGSHT extract had no significant cytotoxicity in preadipocytes or differentiated adipocytes. YGSHT reduced the number of lipid droplets and content of triglyceride in adipose cells. YGSHT also significantly inhibited GPDH activity and decreased leptin production compared with control adipocytes. Down-regulation of peroxisome proliferator-activated receptor-gamma (PPAR-γ) expression at the messenger RNA level was observed in YGSHT-treated adipocytes. Conclusion: Taken together, our data suggest that YGSHT has potential as an anti-obesity drug candidate. PMID:26246724

  20. The inhibition of inflammatory molecule expression on 3T3-L1 adipocytes by berberine is not mediated by leptin signaling.

    PubMed

    Choi, Bong-Hyuk; Kim, Yu-Hee; Ahn, In-Sook; Ha, Jung-Heun; Byun, Jae-Min; Do, Myoung-Sool

    2009-01-01

    In our previous study, we have shown that berberine has both anti-adipogenic and anti-inflammatory effects on 3T3-L1 adipocytes, and the anti-adipogenic effect is due to the down-regulation of adipogenic enzymes and transcription factors. Here we focused more on anti-inflammatory effect of berberine using real time RT-PCR and found it changes expressions of adipokines. We hypothesized that anti-adipogenicity of berberine mediates anti-inflammtory effect and explored leptin as a candidate mediator of this signaling. We studied this hypothesis by western blot analysis, but our results showed that berberine has no effect on the phosphorylations of STAT-3 and ERK which have important roles on leptin signaling. These results led us to conclude that the anti-inflammatory effect of berberine is not mediated by the inhibition of leptin signal transduction. Moreover, we have found that berberine down-regulates NF-kappaB signaling, one of the inflammation-related signaling pathway, through western blot analysis. Taken together, the anti-inflammatory effect of berberine is not mediated by leptin, and berberine induces anti-inflammatory effect independent of leptin signaling.

  1. Glutamine, insulin and glucocorticoids regulate glutamine synthetase expression in C2C12 myotubes, Hep G2 hepatoma cells and 3T3 L1 adipocytes.

    PubMed

    Wang, Yanxin; Watford, Malcolm

    2007-04-01

    The cell-specific regulation of glutamine synthetase expression was studied in three cell lines. In C2C12 myotubes, glucocorticoids increased the abundance of both glutamine synthetase protein and mRNA. Culture in the absence of glutamine also resulted in very high glutamine synthetase protein abundance but mRNA levels were unchanged. Glucocorticoids also increased the abundance of glutamine synthetase mRNA in Hep G2 hepatoma cells but this was not reflected in changes in protein abundance. Culture of Hep G2 cells without glutamine resulted in very high levels of protein, again with no change in mRNA abundance. Insulin was without effect in both C2C12 and Hep G2 cells. In 3T3 L1 adipocytes glucocorticoids increased the abundance of both glutamine synthetase mRNA and protein, insulin added alone had no effect but in the presence of glucocorticoids resulted in lower mRNA levels than seen with glucocorticoids alone, although protein levels remained high under such conditions. In contrast to the other cell lines glutamine synthetase protein levels were relatively unchanged by culture in the absence of glutamine. The results support the hypothesis that in myocytes, and hepatomas, but not in adipocytes, glutamine acts to moderate glutamine synthetase induction by glucocorticoids.

  2. 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.

  3. Cooperation between HMGA1 and HIF-1 Contributes to Hypoxia-Induced VEGF and Visfatin Gene Expression in 3T3-L1 Adipocytes

    PubMed Central

    Messineo, Sebastiano; Laria, Anna Elisa; Arcidiacono, Biagio; Chiefari, Eusebio; Luque Huertas, Raúl M.; Foti, Daniela P.; Brunetti, Antonio

    2016-01-01

    The architectural transcription factor high-mobility group AT-hook 1 (HMGA1) is a chromatin regulator with implications in several biological processes, including tumorigenesis, inflammation, and metabolism. Previous studies have indicated a role for this factor in promoting the early stages of adipogenesis, while inhibiting adipocyte terminal differentiation, and decreasing fat mass. It has been demonstrated that hypoxia – through the hypoxia-inducible factor 1 (HIF-1) – plays a major role in triggering changes in the adipose tissue of the obese, leading to inhibition of adipocyte differentiation, adipose cell dysfunction, inflammation, insulin resistance, and type 2 diabetes. To examine the possible cooperation between HMGA1 and HIF-1, herein, we investigated the role of HMGA1 in the regulation of Visfatin and VEGF, two genes normally expressed in adipose cells, which are both responsive to hypoxia. We demonstrated that HMGA1 enhanced Visfatin and VEGF gene expression in human embryonic kidney (HEK) 293 cells in hypoxic conditions, whereas HMGA1 knockdown in differentiated 3T3-L1 adipocytes reduced these effects. Reporter gene analysis showed that Visfatin and VEGF transcriptional activity was increased by the addition of either HMGA1 or HIF-1 and even further by the combination of both factors. As demonstrated by chromatin immunoprecipitation in intact cells, HMGA1 directly interacted with the VEGF gene, and this interaction was enhanced in hypoxic conditions. Furthermore, as indicated by co-immunoprecipitation studies, HMGA1 and HIF-1 physically interacted with each other, supporting the notion that this association may corroborate a functional link between these factors. Therefore, our findings provide evidence for molecular cross-talk between HMGA1 and HIF-1, and this may be important for elucidating protein and gene networks relevant to obesity. PMID:27445976

  4. PIP3 but not PIP2 increases GLUT4 surface expression and glucose metabolism mediated by AKT/PKCζ/λ phosphorylation in 3T3L1 adipocytes.

    PubMed

    Manna, Prasenjit; Jain, Sushil K

    2013-09-01

    Phosphatidylinositol-3,4,5-triphosphate (PIP3) and phosphatidylinositol-4,5-biphosphate (PIP2) are two well-known membrane bound polyphosphoinositides. Diabetes is associated with impaired glucose metabolism. Using a 3T3L1 adipocyte cell model, this study investigated the role of PIP3 and PIP2 on insulin stimulated glucose metabolism in high glucose (HG) treated cells. Exogenous PIP3 supplementation (1, 5, or 10 nM) increased the phosphorylation of AKT and PKCζ/λ, which in turn upregulated GLUT4 total protein expression as well as its surface expression, glucose uptake, and glucose utilization in cells exposed to HG (25 mM); however, PIP2 had no effect. Comparative signal silencing studies with antisense AKT2 and antisense PKCζ revealed that phosphorylation of PKCζ/λ is more effective in PIP3 mediated GLUT4 activation and glucose utilization than in AKT phosphorylation. Supplementation with PIP3 in combination with insulin enhanced glucose uptake and glucose utilization compared to PIP2 with insulin, or insulin alone, in HG-treated adipocytes. This suggests that a decrease in cellular PIP3 levels may cause impaired insulin sensitivity in diabetes. PIP3 supplementation also prevented HG-induced MCP-1 and resistin secretion and lowered adiponectin levels. This study for the first time demonstrates that PIP3 but not PIP2 plays an important role in GLUT4 upregulation and glucose metabolism mediated by AKT/PKCζ/λ phosphorylation. Whether PIP3 levels in blood can be used as a biomarker of insulin resistance in diabetes needs further investigation.

  5. 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.

  6. Cooling-increased phospho-β-arrestin-1 and β-arrestin-1 expression levels in 3T3-L1 adipocytes.

    PubMed

    Ohsaka, Yasuhito; Nishino, Hoyoku

    2012-08-01

    Cooling induces several responses that are modulated by molecular inhibitors and activators and receptor signaling. Information regarding potential targets involved in cold response mechanisms is still insufficient. We examined levels of the receptor-signaling mediator β-arrestin-1 and phospho-Ser-412 β-arrestin-1 in 3T3-L1 adipocytes exposed to 4-37 °C or treated with some molecular agents at 37°C. We also cooled cells with or without modification and signal-modulating agents. These conditions did not decrease cell viability, and western blot analysis revealed that exposure to 4 °C for 1.5h and to 28 and 32 °C for 24 and 48 h increased phospho-β-arrestin-1 and β-arrestin-1 levels and that exposure to 4 and 18 °C for 3 and 4.5h increased β-arrestin-1 level. Serum removal and rewarming abolished β-arrestin-1 alterations induced by cooling. Mithramycin A (a transcription inhibitor) treatment for 4 and 24h increased the level of β-arrestin-1 but not that of phospho-β-arrestin-1. The level of phospho-β-arrestin-1 was increased by okadaic acid (a phosphatase inhibitor), decreased by epinephrine and aluminum fluoride (receptor-signaling modulators), and unaffected by N-ethylmaleimide (an alkylating agent) at 37 °C. N-Ethylmaleimide and the receptor-signaling modulators did not alter β-arrestin-1 expression at 37 °C but impaired the induction of phospho-β-arrestin-1 at 28 and 32 °C without affecting the induction of β-arrestin-1. We show that cold-induced β-arrestin-1 alterations are partially mimicked by molecular agents and that the responsive machinery for β-arrestin-1 requires serum factors and N-ethylmaleimide-sensitive sites and is linked to rewarming- and receptor signaling-responsive machinery. Our findings provide helpful information for clarifying the cold-responsive machinery for β-arrestin-1 and elucidating low-temperature responses.

  7. Differentiation-specific decrease in heat shock protein synthesis in 3T3-L1 cells

    SciTech Connect

    Sorhage, F.; Kim, J.; Liu, A.Y.C.; Chen, K.Y.

    1986-05-01

    The regulation of synthesis of heat shock proteins (HSPs) in 3T3-L1 preadipocytes (fibroblasts) and adipocytes was examined using the techniques of pulse labeling with (/sup 35/S)methionine followed by analysis of the pattern and amount of radioactivity incorporated by SDS-polyacrylamide gel electrophoresis and autoradiography. Exposure of the 3T3-L1 preadipocyte cultures either to elevated temperature (42..mu..C) or to the amino acid analogue canavanine (400 ..mu..g/ml), markedly induced the synthesis of six major HSPs with apparent molecular weights of 105,000, 89,000, 74,000, 72,000, 50,000, and 42,000. The time course of induction of the HSPs by canavanine was significantly delayed as compared to that of heat shock; maximal increase in synthesis of the HSPs was observed at 3-7 hrs after incubation at 42..mu..c and at 22-24 hrs after incubation with 400 ..mu..g/ml canavanine. The magnitude of induction of HSP in the differentiated adipocytes was significantly reduced as compared to that of the undifferentiated fibroblast cells. The reduced expression of HSPs in 3T3-L1 adipocytes appears to be related to the terminal adipogenic differentiation process. The phenomenon was not observed in the control 3T3-C2 cells nor in a transformed variant of the 3T3-L1 cells.

  8. The effect of hypoxia mimetic cobalt chloride on the expression of EC-SOD in 3T3-L1 adipocytes.

    PubMed

    Kamiya, Tetsuro; Hara, Hirokazu; Inagaki, Naoki; Adachi, Tetsuo

    2010-01-01

    It is well known that adipose tissue is not only a passive reservoir for energy storage but also produces and secretes a variety of bioactive molecules called adipocytokines, including adiponectin and tumor necrosis factor-alpha (TNF-alpha). Recently, it has been reported that adipose tissue can suffer a chronic hypoxic condition during hypertrophy of adipocytes, and this condition leads to the dysregulation of adipocytokines. Further, hypoxic adipocytes are in an increased oxidative stress. Extracellular-superoxide dismutase (EC-SOD) is an anti-inflammatory enzyme that protects cells from reactive oxygen species (ROS) by scavenging superoxide anion. Previous reports showed that plasma EC-SOD levels in type 2 diabetes patients were significantly and inversely related to the body mass index, homeostasis model assessment-insulin resistance index; however, the mechanisms of EC-SOD and adiponectin reductions during hypoxia remain poorly understood. Here, we demonstrate that cobalt chloride (CoCl(2)), a hypoxia mimetic, decreases EC-SOD and adiponectin in 3T3-L1 adipocytes by intracellular ROS-independent, but TNF-alpha and c-jun N-terminal kinase (JNK)-dependent mechanisms. From these results, it is possible that TNF-alpha is a key regulator of the reduction of EC-SOD and adiponectin in CoCl(2)-treated 3T3-L1 adipocytes, and we speculated that the reduction of EC-SOD and adiponectin would lead to and/or promote metabolic disorders.

  9. Molecularly Characterized Solvent Extracts and Saponins from Polygonum hydropiper L. Show High Anti-Angiogenic, Anti-Tumor, Brine Shrimp, and Fibroblast NIH/3T3 Cell Line Cytotoxicity

    PubMed Central

    Ayaz, Muhammad; Junaid, Muhammad; Ullah, Farhat; Sadiq, Abdul; Subhan, Fazal; Khan, Mir Azam; Ahmad, Waqar; Ali, Gowhar; Imran, Muhammad; Ahmad, Sajjad

    2016-01-01

    Polygonum hydropiper is used as anti-cancer and anti-rheumatic agent in folk medicine. This study was designed to investigate the anti-angiogenic, anti-tumor, and cytotoxic potentials of different solvent extracts and isolated saponins. Samples were analyzed using GC, Gas Chromatography–Mass Spectrometry (GC–MS) to identify major and bioactive compounds. Quantitation of antiangiogenesis for the plant's samples including methanolic extract (Ph.Cr), its subsequent fractions; n-hexane (Ph.Hex), chloroform (Ph.Chf), ethyl acetate (Ph.EtAc), n-Butanol (Ph.Bt), aqueous (Ph.Aq), saponins (Ph.Sp) were performed using the chick embryo chorioallantoic membrane (CAM) assay. Potato disc anti-tumor assay was performed on Agrobacterium tumefaciens containing tumor inducing plasmid. Cytotoxicity was performed against Artemia salina and mouse embryonic fibroblast NIH/3T3 cell line following contact toxicity and MTT cells viability assays, respectively. The GC–MS analysis of Ph.Cr, Ph.Hex, Ph.Chf, Ph.Bt, and Ph.EtAc identified 126, 124, 153, 131, and 164 compounds, respectively. In anti-angiogenic assay, Ph.Chf, Ph.Sp, Ph.EtAc, and Ph.Cr exhibited highest activity with IC50 of 28.65, 19.21, 88.75, and 461.53 μg/ml, respectively. In anti-tumor assay, Ph.Sp, Ph.Chf, Ph.EtAc, and Ph.Cr were most potent with IC50 of 18.39, 73.81, 217.19, and 342.53 μg/ml, respectively. In MTT cells viability assay, Ph.Chf, Ph.EtAc, Ph.Sp were most active causing 79.00, 72.50, and 71.50% cytotoxicity, respectively, at 1000 μg/ml with the LD50 of 140, 160, and 175 μg/ml, respectively. In overall study, Ph.Chf and Ph.Sp have shown overwhelming results which signifies their potentials as sources of therapeutic agents against cancer. PMID:27065865

  10. Ethanol extracts of chickpeas alter the total lipid content and expression levels of genes related to fatty acid metabolism in mouse 3T3-L1 adipocytes.

    PubMed

    Shinohara, Shigeo; Gu, Yuanjun; Yang, Ying; Furuta, Yasuo; Tanaka, Masahiko; Yue, Xiaohua; Wang, Weiqing; Kitano, Masaru; Kimura, Hiroshi

    2016-08-01

    Desi-type chickpeas, which have long been used as a natural treatment for diabetes, have been reported to lower visceral adiposity, dyslipidemia and insulin resistance induced by a chronic high-fat diet in rats. In this study, in order to examine the effects of chickpeas of this type in an in vitro system, we used the 3T3-L1 mouse cell line, a subclone of Swiss 3T3 cells, which can differentiate into cells with an adipocyte-like phenotype, and we used ethanol extracts of chickpeas (ECP) instead of chickpeas. Treatment of the 3T3-L1 cells with ECP led to a decrease in the lipid content in the cells. The desaturation index, defined as monounsaturated fatty acids (MUFAs)/saturated fatty acids (SFAs), was also decreased by ECP due to an increase in the cellular content of SFAs and a decrease in the content of MUFAs. The decrease in this index may reflect a decreased reaction from SFA to MUFA, which is essential for fat storage. To confirm this hypothesis, we conducted a western blot analysis, which revealed a reduction in the amount of stearoyl-CoA desaturase 1 (SCD1), a key enzyme catalyzing the reaction from SFA to MUFA. We observed simultaneous inactivations of enzymes participating in lipogenesis, i.e., liver kinase B1 (LKB1), acetyl-CoA carboxylase (ACC) and AMPK, by phosphorylation, which may lead to the suppression of reactions from acetyl-CoA to SFA via malonyl-CoA in lipogenesis. We also investigated whether lipolysis is affected by ECP. The amount of carnitine palmitoyltransferase 1 (CPT1), an enzyme important for the oxidation of fatty acids, was increased by ECP treatment. ECP also led to an increase in uncoupling protein 2 (UCP2), reported as a key protein for the oxidation of fatty acids. All of these results obtained regarding lipogenesis and fatty acid metabolism in our in vitro system are consistent with the results previously shown in rats. We also examined the effects on SCD1 and lipid contents of ethanol extracts of Kabuli

  11. Ethanol extracts of chickpeas alter the total lipid content and expression levels of genes related to fatty acid metabolism in mouse 3T3-L1 adipocytes

    PubMed Central

    Shinohara, Shigeo; Gu, Yuanjun; Yang, Ying; Furuta, Yasuo; Tanaka, Masahiko; Yue, Xiaohua; Wang, Weiqing; Kitano, Masaru; Kimura, Hiroshi

    2016-01-01

    Desi-type chickpeas, which have long been used as a natural treatment for diabetes, have been reported to lower visceral adiposity, dyslipidemia and insulin resistance induced by a chronic high-fat diet in rats. In this study, in order to examine the effects of chickpeas of this type in an in vitro system, we used the 3T3-L1 mouse cell line, a subclone of Swiss 3T3 cells, which can differentiate into cells with an adipocyte-like phenotype, and we used ethanol extracts of chickpeas (ECP) instead of chickpeas. Treatment of the 3T3-L1 cells with ECP led to a decrease in the lipid content in the cells. The desaturation index, defined as monounsaturated fatty acids (MUFAs)/saturated fatty acids (SFAs), was also decreased by ECP due to an increase in the cellular content of SFAs and a decrease in the content of MUFAs. The decrease in this index may reflect a decreased reaction from SFA to MUFA, which is essential for fat storage. To confirm this hypothesis, we conducted a western blot analysis, which revealed a reduction in the amount of stearoyl-CoA desaturase 1 (SCD1), a key enzyme catalyzing the reaction from SFA to MUFA. We observed simultaneous inactivations of enzymes participating in lipogenesis, i.e., liver kinase B1 (LKB1), acetyl-CoA carboxylase (ACC) and AMPK, by phosphorylation, which may lead to the suppression of reactions from acetyl-CoA to SFA via malonyl-CoA in lipogenesis. We also investigated whether lipolysis is affected by ECP. The amount of carnitine palmitoyltransferase 1 (CPT1), an enzyme important for the oxidation of fatty acids, was increased by ECP treatment. ECP also led to an increase in uncoupling protein 2 (UCP2), reported as a key protein for the oxidation of fatty acids. All of these results obtained regarding lipogenesis and fatty acid metabolism in our in vitro system are consistent with the results previously shown in rats. We also examined the effects on SCD1 and lipid contents of ethanol extracts of Kabuli-type chickpeas, which are

  12. Characterization of the pharmacology, signal transduction and internalization of the fluorescent PACAP ligand, fluor-PACAP, on NIH/3T3 cells expressing PAC1.

    PubMed

    Germano, P M; Stalter, J; Le, S V; Wu, M; Yamaguchi, D J; Scott, D; Pisegna, J R

    2001-06-01

    Fluor-PACAP, a fluorescent derivative of PACAP-27, has been confirmed to share a high affinity for PAC1 receptors transfected into NIH/3T3 cells and to have comparable pharmacological characteristics to the unconjugated, native form. Through competitive binding with 125I-PACAP-27, the two ligands exhibited similar dose- dependent inhibition. Additional examination of the efficacy of activating adenylyl cyclase revealed that both ligands analogously stimulated the production of cyclic AMP. Furthermore, PAC1 internalization visualized by our Fluor-PACAP, is compareable to that performed with the radioligand, 125I-PACAP-27, with maximal internalization achieved within thirty minutes. Thus, Fluor-PACAP exhibits intracellular signaling abilities homologous to the native ligand.

  13. Disruption of Lipid Raft Function Increases Expression and Secretion of Monocyte Chemoattractant Protein-1 in 3T3-L1 Adipocytes

    PubMed Central

    Lin, Yu-Chun; Chang, Yu-Tzu; Lu, Chia-Yun; Chen, Tzu-Yu; Yeh, Chia-Shan

    2016-01-01

    The adipocyte is unique in its capacity to store lipids. In addition to triglycerides, the adipocyte stores a significant amount of cholesterol. Moreover, obese adipocytes are characterized by a redistribution of cholesterol with depleted cholesterol in the plasma membrane, suggesting that cholesterol perturbation may play a role in adipocyte dysfunction. We used methyl-β-cyclodextrin (MβCD), a molecule with high affinity for cholesterol, to rapidly deplete cholesterol level in differentiated 3T3-L1 adipocytes. We tested whether this perturbation altered adipocyte secretion of monocyte chemoattractant protein-1 (MCP-1), a chemokine that is elevated in obesity and is linked to obesity-associated chronic diseases. Depletion of cholesterol by MβCD increased MCP-1 secretion as well as the mRNA and protein levels, suggesting perturbation at biosynthesis and secretion. Pharmacological inhibition revealed that NF-κB, but not MEK, p38 and JNK, was involved in MβCD-stimulated MCP-1 biosynthesis and secretion in adipocytes. Finally, another cholesterol-binding drug, filipin, also induced MCP-1 secretion without altering membrane cholesterol level. Interestingly, both MβCD and filipin disturbed the integrity of lipid rafts, the membrane microdomains enriched in cholesterol. Thus, the depletion of membrane cholesterol in obese adipocytes may result in dysfunction of lipid rafts, leading to the elevation of proinflammatory signaling and MCP-1 secretion in adipocytes. PMID:28030645

  14. Definition of metabolism-dependent xenobiotic toxicity with co-cultures of human hepatocytes and mouse 3T3 fibroblasts in the novel integrated discrete multiple organ co-culture (IdMOC) experimental system: results with model toxicants aflatoxin B1, cyclophosphamide and tamoxifen.

    PubMed

    Li, Albert P; Uzgare, Aarti; LaForge, Yumiko S

    2012-07-30

    The integrated discrete multiple organ co-culture system (IdMOC) allows the co-culturing of multiple cell types as physically separated cells interconnected by a common overlying medium. We report here the application of IdMOC with two cell types: the metabolically competent primary human hepatocytes, and a metabolically incompetent cell line, mouse 3T3 fibroblasts, in the definition of the role of hepatic metabolism on the cytotoxicity of three model toxicants: cyclophosphamide (CPA), aflatoxin B1 (AFB) and tamoxifen (TMX). The presence of hepatic metabolism in IdMOC with human hepatocytes was demonstrated by the metabolism of the P450 isoform 3A4 substrate, luciferin-IPA. The three model toxicants showed three distinct patterns of cytotoxic profile: TMX was cytotoxic to 3T3 cells in the absence of hepatocytes, with slightly lower cytotoxicity towards both 3T3 cells and hepatocytes in the IdMOC. AFB was selective toxic towards the human hepatocytes and relatively noncytotoxic towards 3T3 cells both in the presence and absence of the hepatocytes. CPA cytotoxicity to the 3T3 cells was found to be significantly enhanced by the presence of the hepatocytes, with the cytotoxicity dependent of the number of hepatocytes, and with the cytotoxicity attenuated by the presence of a non-specific P450 inhibitor, 1-aminobenzotriazole. We propose here the following classification of toxicants based on the role of hepatic metabolism as defined by the human hepatocyte-3T3 cell IdMOC assay: type I: direct-acting cytotoxicants represented by TMX as indicated by cytotoxicity in 3T3 cells in the absence of hepatocytes; type II: metabolism-dependent cytotoxicity represented by AFB1 with effects localized within the site of metabolic activation (i. e. hepatocytes); and type III: metabolism-dependent cytotoxicity with metabolites that can diffuse out of the hepatocytes to cause toxicity in cells distal from the site of metabolism, as exemplified by CPA.

  15. Sida rhomboidea. Roxb leaf extract down-regulates expression of PPARγ2 and leptin genes in high fat diet fed C57BL/6J Mice and retards in vitro 3T3L1 pre-adipocyte differentiation.

    PubMed

    Thounaojam, Menaka C; Jadeja, Ravirajsinh N; Ramani, Umed V; Devkar, Ranjitsinh V; Ramachandran, A V

    2011-01-01

    Sida rhomboidea. Roxb leaf extract (SRLE) is being used by the populace of North-East India to alleviate symptoms of diabetes and obesity. We have previously reported its hypolipidemic and anti-diabetic properties. In this study, we report the effect of SRLE on (i) in vivo modulation of genes controlling high fat diet (HFD) induced obesity and (ii) in vitro 3T3L1 pre-adipocyte differentiation and leptin release. Supplementation with SRLE significantly prevented HFD induced increment in bodyweight, plasma lipids and leptin, visceral adiposity and adipocyte hypertrophy. Also, SRLE supplementation reduced food intake, down regulated PPARγ2, SREBP1c, FAS and LEP expressions and up-regulated CPT-1 in epididymal adipose tissue compared to obese mice. In vitro adipogenesis of 3T3L1 pre-adipocytes was significantly retarded in the presence of SRLE extract. Also decreased triglyceride accumulation, leptin release and glyceraldehyde-3-Phosphate dehydrogenase activity along with higher glycerol release without significant alteration of viability of 3T3L1 pre-adipocytes, was recorded. Our findings suggest that prevention of HFD induced visceral adiposity is primarily by down regulation of PPARγ2 and leptin gene expression coupled with attenuation of food intake in C57BL/6J mice. SRLE induced prevention of pre-adipocytes differentiation, and leptin release further substantiated these findings and scientifically validates the potential application of SRLE as a therapeutic agent against obesity.

  16. C-reactive protein inhibits high-molecular-weight adiponectin expression in 3T3-L1 adipocytes via PI3K/Akt pathway.

    PubMed

    Liu, Yuanxin; Liu, Cuiping; Jiang, Chao; Wang, Su; Yang, Qichao; Jiang, Dan; Yuan, Guoyue

    2016-03-25

    Adiponectin, an adipose-specific protein hormone, is secreted from white adipose tissue and involved in glucose and lipid metabolism. It is assembled into low-molecular-weight trimer (LMW), middle-molecular-weight hexameric (MMW) and high-molecular-weight (HMW), among which HMW exhibits higher activity. In this study, we proved that C-reactive protein (CRP), an inflammatory marker, inhibited adiponectin expression, especially HMW in time-and dose-dependent manners. Furthermore, CRP decreased the HMW/total adiponectin ration and reduced adiponectin assembly by increasing ERp44, and decreasing Ero1-α and DsbA-L. CRP activated pAkt, the downstream of PI3K. Inhibition of PI3K or pAkt abolished the effect of CRP. Our study suggested that CRP decreased adiponectin expression and multimerization, while CRP-induced decline in adiponectin might be mediated through the PI3K/Akt pathway.

  17. 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.

  18. Synthesis, Characterization, and Study of In Vitro Cytotoxicity of ZnO-Fe3O4 Magnetic Composite Nanoparticles in Human Breast Cancer Cell Line (MDA-MB-231) and Mouse Fibroblast (NIH 3T3).

    PubMed

    Bisht, Gunjan; Rayamajhi, Sagar; Kc, Biplab; Paudel, Siddhi Nath; Karna, Deepak; Shrestha, Bhupal G

    2016-12-01

    Novel magnetic composite nanoparticles (MCPs) were successfully synthesized by ex situ conjugation of synthesized ZnO nanoparticles (ZnO NPs) and Fe3O4 NPs using trisodium citrate as linker with an aim to retain key properties of both NPs viz. inherent selectivity towards cancerous cell and superparamagnetic nature, respectively, on a single system. Successful characterization of synthesized nanoparticles was done by XRD, TEM, FTIR, and VSM analyses. VSM analysis showed similar magnetic profile of thus obtained MCPs as that of naked Fe3O4 NPs with reduction in saturation magnetization to 16.63 emu/g. Also, cell viability inferred from MTT assay showed that MCPs have no significant toxicity towards noncancerous NIH 3T3 cells but impart significant toxicity at similar concentration to breast cancer cell MDA-MB-231. The EC50 value of MCPs on MDA-MB-231 is less than that of naked ZnO NPs on MDA-MB-231, but its toxicity on NIH 3T3 was significantly reduced compared to ZnO NPs. Our hypothesis for this prominent difference in cytotoxicity imparted by MCPs is the synergy of selective cytotoxicity of ZnO nanoparticles via reactive oxygen species (ROS) and exhausting scavenging activity of cancerous cells, which further enhance the cytotoxicity of Fe3O4 NPs on cancer cells. This dramatic difference in cytotoxicity shown by the conjugation of magnetic Fe3O4 NPs with ZnO NPs should be further studied that might hold great promise for the development of selective and site-specific nanoparticles. Schematic representation of the conjugation, characterization and cytotoxicity analysis of Fe3O4-ZnO magnetic composite particles (MCPs).

  19. Synthesis, Characterization, and Study of In Vitro Cytotoxicity of ZnO-Fe3O4 Magnetic Composite Nanoparticles in Human Breast Cancer Cell Line (MDA-MB-231) and Mouse Fibroblast (NIH 3T3)

    NASA Astrophysics Data System (ADS)

    Bisht, Gunjan; Rayamajhi, Sagar; KC, Biplab; Paudel, Siddhi Nath; Karna, Deepak; Shrestha, Bhupal G.

    2016-12-01

    Novel magnetic composite nanoparticles (MCPs) were successfully synthesized by ex situ conjugation of synthesized ZnO nanoparticles (ZnO NPs) and Fe3O4 NPs using trisodium citrate as linker with an aim to retain key properties of both NPs viz. inherent selectivity towards cancerous cell and superparamagnetic nature, respectively, on a single system. Successful characterization of synthesized nanoparticles was done by XRD, TEM, FTIR, and VSM analyses. VSM analysis showed similar magnetic profile of thus obtained MCPs as that of naked Fe3O4 NPs with reduction in saturation magnetization to 16.63 emu/g. Also, cell viability inferred from MTT assay showed that MCPs have no significant toxicity towards noncancerous NIH 3T3 cells but impart significant toxicity at similar concentration to breast cancer cell MDA-MB-231. The EC50 value of MCPs on MDA-MB-231 is less than that of naked ZnO NPs on MDA-MB-231, but its toxicity on NIH 3T3 was significantly reduced compared to ZnO NPs. Our hypothesis for this prominent difference in cytotoxicity imparted by MCPs is the synergy of selective cytotoxicity of ZnO nanoparticles via reactive oxygen species (ROS) and exhausting scavenging activity of cancerous cells, which further enhance the cytotoxicity of Fe3O4 NPs on cancer cells. This dramatic difference in cytotoxicity shown by the conjugation of magnetic Fe3O4 NPs with ZnO NPs should be further studied that might hold great promise for the development of selective and site-specific nanoparticles.

  20. Fisetin up-regulates the expression of adiponectin in 3T3-L1 adipocytes via the activation of silent mating type information regulation 2 homologue 1 (SIRT1)-deacetylase and peroxisome proliferator-activated receptors (PPARs).

    PubMed

    Jin, Taewon; Kim, Oh Yoen; Shin, Min-Jeong; Choi, Eun Young; Lee, Sung Sook; Han, Ye Sun; Chung, Ji Hyung

    2014-10-29

    Adiponectin, an adipokine, has been described as showing physiological benefits against obesity-related malfunctions and vascular dysfunction. Several natural compounds that promote the expression and secretion of adipokines in adipocytes could be useful for treating metabolic disorders. This study investigated the effect of fisetin, a dietary flavonoid, on the regulation of adiponectin in adipocytes using 3T3-L1 preadipocytes. The expression and secretion of adiponectin increased in 3T3-L1 cells upon treatment with fisetin in a dose-dependent manner. Fisetin-induced adiponectin secretion was inhibited by peroxisome proliferator-activated receptor (PPAR) antagonists. It was also revealed that fisetin increased the activities of PPARs and silent mating type information regulation 2 homologue 1 (SIRT1) in a dose-dependent manner. Furthermore, the up-regulation of adiponectin and the activation of PPARs induced by fisetin were prevented by a SIRT1 inhibitor. Fisetin also promoted deacetylation of PPAR γ coactivator 1 (PGC-1) and its interaction with PPARs. SIRT knockdown by siRNA significantly decreased both adiponectin production and PPARs-PGC-1 interaction. These results provide evidence that fisetin promotes the gene expression of adiponectin through the activation of SIRT1 and PPARs in adipocytes.

  1. 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.

  2. Mouse white adipocytes and 3T3-L1 cells display an anomalous pattern of carnitine palmitoyltransferase (CPT) I isoform expression during differentiation. Inter-tissue and inter-species expression of CPT I and CPT II enzymes.

    PubMed Central

    Brown, N F; Hill, J K; Esser, V; Kirkland, J L; Corkey, B E; Foster, D W; McGarry, J D

    1997-01-01

    The outer mitochondrial membrane enzyme carnitine palmitoyltransferase I (CPT I) represents the initial and regulated step in the beta-oxidation of fatty acids. It exists in at least two isoforms, denoted L (liver) and M (muscle) types, with very different kinetic properties and sensitivities to malonyl-CoA. Here we have examined the relative expression of the CPT I isoforms in two different models of adipocyte differentiation and in a number of rat tissues. Adipocytes from mice, hamsters and humans were also evaluated. Primary monolayer cultures of undifferentiated rat preadipocytes expressed solely L-CPT I, but significant levels of M-CPT I emerged after only 3 days of differentiation in vitro; in the mature cell M-CPT I predominated. In sharp contrast, the murine 3T3-L1 preadipocyte expressed essentially exclusively L-CPT I, both in the undifferentiated state and throughout the differentiation process in vitro. This was also true of the mature mouse white fat cell. Fully developed adipocytes from the hamster and human behaved similarly to those of the rat. Thus the mouse white fat cell differs fundamentally from those of the other species examined in terms of tis choice of a key regulatory enzyme in fatty acid metabolism. In contrast, brown adipose tissue from all three rodents displayed the same isoform profiles, each expressing overwhelmingly M-CPT I. Northern blot analysis of other rat tissues established L-CPT I as the dominant isoform not only in liver but also in kidney, lung, ovary, spleen, brain, intestine and pancreatic islets. In addition to its primacy in skeletal muscle, heart and fat, M-CPT I was also found to dominate the testis. The same inter-tissue isoform pattern (with the exception of white fat) was found in the mouse. Taken together, the data bring to light an intriguing divergence between white adipocytes of the mouse and other mammalian species. They also raise a cautionary note that should be considered in the choice of animal model used

  3. Troglitazone inhibits the expression of inducible nitric oxide synthase in adipocytes in vitro and in vivo study in 3T3-L1 cells and Otsuka Long-Evans Tokushima Fatty rats.

    PubMed

    Dobashi, K; Asayama, K; Nakane, T; Kodera, K; Hayashibe, H; Nakazawa, S

    2000-09-15

    The aim of this study was to determine the mechanism of troglitazone action on nitric oxide (NO) production via inducible NO synthase (iNOS) in adipocytes in vitro and in vivo. The treatment of 3T3-L1 adipocytes with the combination of lipopolysaccharide (LPS), tumor necrosis factor-alpha and interferon-gamma synergistically induced de novo iNOS expression leading to enhanced NO production. The NO production was inhibited by co-treatment with aminoguanidine or N-nitro-L-arginine methylester hydrochloride. Troglitazone inhibited the NO production in a dose dependent manner by the suppression of iNOS expression. In the 24 week-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats, the mean weight and the blood glucose were 21% and 30%, respectively, higher than in their lean counterparts. The serum nitrite concentration was increased after injection of LPS (4 mg/kg, i.p.), more markedly in OLETF rats than in the lean rats. The epididymal fats from LPS-injected groups, but not the ones from the non-injected groups, expressed mRNA and protein of iNOS. Troglitazone pre-treatment blocked the LPS-induced expression of iNOS in adipose tissue and the increase in serum nitrite concentration. These results suggest that troglitazone inhibits the cytokine-induced NO production in adipocytes by blocking iNOS expression both in vitro and in vivo.

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

    PubMed

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

    1990-01-01

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

  5. Combined Effects of Androgen and Growth Hormone on Osteoblast Marker Expression in Mouse C2C12 and MC3T3-E1 Cells Induced by Bone Morphogenetic Protein

    PubMed Central

    Kimura, Kosuke; Terasaka, Tomohiro; Iwata, Nahoko; Katsuyama, Takayuki; Komatsubara, Motoshi; Nagao, Ryota; Inagaki, Kenichi; Otsuka, Fumio

    2017-01-01

    Osteoblasts undergo differentiation in response to various factors, including growth factors and steroids. Bone mass is diminished in androgen- and/or growth hormone (GH)-deficient patients. However the functional relationship between androgen and GH, and their combined effects on bone metabolism, remains unclear. Here we investigated the mutual effects of androgen and GH on osteoblastic marker expression using mouse myoblastic C2C12 and osteoblast-like MC3T3-E1 cells. Combined treatment with dihydrotestosterone (DHT) and GH enhanced BMP-2-induced expression of Runx2, ALP, and osteocalcin mRNA, compared with the individual treatments in C2C12 cells. Co-treatment with DHT and GH activated Smad1/5/8 phosphorylation, Id-1 transcription, and ALP activity induced by BMP-2 in C2C12 cells but not in MC3T3-E1 cells. The insulin-like growth factor (IGF-I) mRNA level was amplified by GH and BMP-2 treatment and was restored by co-treatment with DHT in C2C12 cells. The mRNA level of the IGF-I receptor was not significantly altered by GH or DHT, while it was increased by IGF-I. In addition, IGF-I treatment increased collagen-1 mRNA expression, whereas blockage of endogenous IGF-I activity using an anti-IGF-I antibody failed to suppress the effect of GH and DHT on BMP-2-induced Runx2 expression in C2C12 cells, suggesting that endogenous IGF-I was not substantially involved in the underlying GH actions. On the other hand, androgen receptor and GH receptor mRNA expression was suppressed by BMP-2 in both cell lines, implying the existence of a feedback action. Collectively the results showed that the combined effects of androgen and GH facilitated BMP-2-induced osteoblast differentiation at an early stage by upregulating BMP receptor signaling. PMID:28067796

  6. Characterization of the growth of murine fibroblasts that express human insulin receptors. II. Interaction of insulin with other growth factors

    SciTech Connect

    Randazzo, P.A.; Jarett, L. )

    1990-09-01

    The effects of insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and insulin on DNA synthesis were studied in murine fibroblasts transfected with an expression vector containing human insulin receptor cDNA (NIH 3T3/HIR) and the parental NIH 3T3 cells. In NIH 3T3/HIR cells, individual growth factors in serum-free medium stimulated DNA synthesis with the following relative efficacies: insulin greater than or equal to 10% fetal calf serum greater than PDGF greater than IGF-1 much greater than EGF. In comparison, the relative efficacies of these factors in stimulating DNA synthesis by NIH 3T3 cells were 10% fetal calf serum greater than PDGF greater than EGF much greater than IGF-1 = insulin. In NIH 3T3/HIR cells, EGF was synergistic with 1-10 ng/ml insulin but not with 100 ng/ml insulin or more. Synergy of PDGF or IGF-1 with insulin was not detected. In the parental NIH 3T3 cells, insulin and IGF-1 were found to be synergistic with EGF (1 ng/ml), PDGF (100 ng/ml), and PDGF plus EGF. In NIH 3T3/HIR cells, the lack of interaction of insulin with other growth factors was also observed when the percentage of cells synthesizing DNA was examined. Despite insulin's inducing only 60% of NIH 3T3/HIR cells to incorporate thymidine, addition of PDGF, EGF, or PDGF plus EGF had no further effect. In contrast, combinations of growth factors resulted in 95% of the parental NIH 3T3 cells synthesizing DNA. The independence of insulin-stimulated DNA synthesis from other mitogens in the NIH 3T3/HIR cells is atypical for progression factor-stimulated DNA synthesis and is thought to be partly the result of insulin receptor expression in an inappropriate context or quantity.

  7. Glabridin Alleviates the Toxic Effects of Methylglyoxal on Osteoblastic MC3T3-E1 Cells by Increasing Expression of the Glyoxalase System and Nrf2/HO-1 Signaling and Protecting Mitochondrial Function.

    PubMed

    Choi, Eun Mi; Suh, Kwang Sik; Kim, Yu Jin; Hong, Soo Min; Park, So Yong; Chon, Suk

    2016-01-13

    Methylglyoxal (MG) contributes to the pathogenesis of age- and diabetes-associated complications. The present study investigated the effects of glabridin on MG-induced cytotoxicity in MC3T3-E1 osteoblastic cells. MC3T3-E1 cells were treated with glabridin in the presence of MG, and markers of mitochondrial function and oxidative damage were examined. Pretreatment of MC3T3-E1 osteoblastic cells with glabridin prevented MG-induced cell death, the production of intracellular reactive oxygen species and mitochondrial superoxides, cardiolipin peroxidation, and the production of inflammatory cytokines. The soluble form of receptor for advanced glycation end products (sRAGEs)/RAGE ratio increased upon MG treatment, but less so after pretreatment with glabridin, which also increased the level of reduced glutathione and the activities of glyoxalase I and heme oxygenase-1, all of which were reduced by MG. In addition, glabridin elevated the level of nuclear factor erythroid 2-related factor 2. These findings suggest that glabridin protects against MG-induced cell damage by inhibiting oxidative stress and increasing MG detoxification. Pretreatment of MC3T3-E1 osteoblastic cells with glabridin reduced MG-induced mitochondrial dysfunction. Additionally, the nitric oxide level significantly increased upon glabridin pretreatment. Together, these data show that glabridin may potentially serve to prevent the development of diabetic bone disease associated with MG-induced oxidative stress.

  8. Cannabidiol promotes browning in 3T3-L1 adipocytes.

    PubMed

    Parray, Hilal Ahmad; Yun, Jong Won

    2016-05-01

    Recruitment of the brown-like phenotype in white adipocytes (browning) and activation of existing brown adipocytes are currently being investigated as a means to combat obesity. Thus, a wide variety of dietary agents that contribute to browning of white adipocytes have been identified. The present study was designed to investigate the effects of cannabidiol (CBD), a major nonpsychotropic phytocannabinoid of Cannabis sativa, on induction of browning in 3T3-L1 adipocytes. CBD enhanced expression of a core set of brown fat-specific marker genes (Ucp1, Cited1, Tmem26, Prdm16, Cidea, Tbx1, Fgf21, and Pgc-1α) and proteins (UCP1, PRDM16, and PGC-1α). Increased expression of UCP1 and other brown fat-specific markers contributed to the browning of 3T3-L1 adipocytes possibly via activation of PPARγ and PI3K. In addition, CBD increased protein expression levels of CPT1, ACSL, SIRT1, and PLIN while down-regulating JNK2, SREBP1, and LPL. These data suggest possible roles for CBD in browning of white adipocytes, augmentation of lipolysis, thermogenesis, and reduction of lipogenesis. In conclusion, the current data suggest that CBD plays dual modulatory roles in the form of inducing the brown-like phenotype as well as promoting lipid metabolism. Thus, CBD may be explored as a potentially promising therapeutic agent for the prevention of obesity.

  9. Gene expression profile of mouse fibroblasts exposed to a biodegradable iron alloy for stents.

    PubMed

    Purnama, Agung; Hermawan, Hendra; Champetier, Serge; Mantovani, Diego; Couet, Jacques

    2013-11-01

    Iron-based materials could constitute an interesting option for cardiovascular biodegradable stent applications due to their superior ductility compared to their counterparts - magnesium alloys. Since the predicted degradation rate of pure iron is considered slow, manganese (35% w/w), an alloying element for iron, was explored to counteract this problem through the powder metallurgy process (Fe-35 Mn). However, manganese presents a high cytotoxic potential; thus its effect on cells must first be established. Here, we established the gene expression profile of mouse 3T3 fibroblasts exposed to Fe-35 Mn degradation products in order to better understand cell response to potentially cytotoxic degradable metallic material (DMM). Mouse 3T3 cells were exposed to degradation products eluting through tissue culture insert filter (3 μm pore size) containing cytostatic amounts of 3.25 mg ml(-1) of Fe-35 Mn powder, 0.25 mg ml(-1) of pure Mn powder or 5 mg ml(-1) of pure iron powder for 24 h. We then conducted a gene expression profiling study from these cells. Exposure of 3T3 cells to Fe-35 Mn was associated with the up-regulation of 75 genes and down-regulation of 59 genes, while 126 were up-regulated and 76 down-regulated genes in the presence of manganese. No genes were found regulated for the iron powder. When comparing the GEP of 3T3 fibroblasts in the presence of Fe-35 Mn and Mn, 68 up-regulated and 54 down-regulated genes were common. These results were confirmed by quantitative RT-PCR for a subset of these genes. This GEP study could provide clues about the mechanism behind degradation products effects on cells of the Fe-35 Mn alloy and may help in the appraisal of its potential for DMM applications.

  10. 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/β-catenin signaling pathway.

    PubMed

    Kim, Young-Min; Kim, In-Hye; Choi, Jeong-Wook; Lee, Min-Kyeong; Nam, Taek-Jeong

    2015-08-01

    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-α expression, which in turn activated various subfactors. An upstream effector of glycogen synthase kinase-3β (GSK-3β) 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/β-catenin signaling pathway.

  11. Mechanoregulation of gene expression in fibroblasts

    PubMed Central

    Wang, James H.-C.; Thampatty, Bhavani P.; Lin, Jeen-Shang; Im, Hee-Jeong

    2010-01-01

    Mechanical loads placed on connective tissues alter gene expression in fibroblasts through mechanotransduction mechanisms by which cells convert mechanical signals into cellular biological events, such as gene expression of extracellular matrix components (e.g., collagen). This mechanical regulation of ECM gene expression affords maintenance of connective tissue homeostasis. However, mechanical loads can also interfere with homeostatic cellular gene expression and consequently cause the pathogenesis of connective tissue diseases such as tendinopathy and osteoarthritis. Therefore, the regulation of gene expression by mechanical loads is closely related to connective tissue physiology and pathology. This article reviews the effects of various mechanical loading conditions on gene regulation in fibroblasts and discusses several mechanotransduction mechanisms. Future research directions in mechanoregulation of gene expression are also suggested. PMID:17331678

  12. Aspartame downregulates 3T3-L1 differentiation.

    PubMed

    Pandurangan, Muthuraman; Park, Jeongeun; Kim, Eunjung

    2014-10-01

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

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

    SciTech Connect

    Lai, Peng-Yeh; Tsai, Chong-Bin; 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/EBPδ and PPARγ 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/EBPα, PPARγ, aP2, LPL and HSL during the middle and late stages of 3T3-L1 adipogenesis.

  14. Increased Oxidative Stress in Cultured 3T3-L1 Cells was Attenuated by Berberine Treatment.

    PubMed

    Dong, Shi-Fen; Yasui, Naomi; Negishb, Hiroko; Kishimoto, Aya; Sun, Jian-Ning; Ikeda, Katsumi

    2015-06-01

    The 3T3-L1 cell line is one of the most well-characterized and reliable models for studying adipocytes. Increased oxidative stress in accumulated fat was found in 3T3-L1 cells. Berberine, an isoquinoline alkaloid, could suppress fat deposition in 3T3-L1 cells; however, whether berberine suppresses increased oxidative stress is not well known. In this study, we observed the effect of berberine on increased oxidative stress in 3T3-L1 cells. 3T3-L1 cells were cultured and treated with berberine (5-20 μM) from day 3 to day 8. We confirmed that berberine markedly inhibited fat accumulation and lipid droplets in 3T3-L1 adipocytes and decreased triglyceride content. Berberine inhibited increased oxidative stress in 3T3-L1 cells by suppressing reactive oxygen species (ROS) production, and increased glutathione peroxidase (GPx) gene expression and GPx activity. Berberine also markedly reduced adipokines secreted by adipocytes, including leptin and resistin.

  15. Gastrointestinal Fibroblasts Have Specialized, Diverse Transcriptional Phenotypes: A Comprehensive Gene Expression Analysis of Human Fibroblasts

    PubMed Central

    Ishii, Genichiro; Aoyagi, Kazuhiko; Sasaki, Hiroki; Ochiai, Atsushi

    2015-01-01

    Background Fibroblasts are the principal stromal cells that exist in whole organs and play vital roles in many biological processes. Although the functional diversity of fibroblasts has been estimated, a comprehensive analysis of fibroblasts from the whole body has not been performed and their transcriptional diversity has not been sufficiently explored. The aim of this study was to elucidate the transcriptional diversity of human fibroblasts within the whole body. Methods Global gene expression analysis was performed on 63 human primary fibroblasts from 13 organs. Of these, 32 fibroblasts from gastrointestinal organs (gastrointestinal fibroblasts: GIFs) were obtained from a pair of 2 anatomical sites: the submucosal layer (submucosal fibroblasts: SMFs) and the subperitoneal layer (subperitoneal fibroblasts: SPFs). Using hierarchical clustering analysis, we elucidated identifiable subgroups of fibroblasts and analyzed the transcriptional character of each subgroup. Results In unsupervised clustering, 2 major clusters that separate GIFs and non-GIFs were observed. Organ- and anatomical site-dependent clusters within GIFs were also observed. The signature genes that discriminated GIFs from non-GIFs, SMFs from SPFs, and the fibroblasts of one organ from another organ consisted of genes associated with transcriptional regulation, signaling ligands, and extracellular matrix remodeling. Conclusions GIFs are characteristic fibroblasts with specific gene expressions from transcriptional regulation, signaling ligands, and extracellular matrix remodeling related genes. In addition, the anatomical site- and organ-dependent diversity of GIFs was also discovered. These features of GIFs contribute to their specific physiological function and homeostatic maintenance, and create a functional diversity of the gastrointestinal tract. PMID:26046848

  16. Mitomycin C modulates the circadian oscillation of clock gene period 2 expression through attenuating the glucocorticoid signaling in mouse fibroblasts.

    PubMed

    Kusunose, Naoki; Matsunaga, Naoya; Kimoto, Kenichi; Akamine, Takahiro; Hamamura, Kengo; Koyanagi, Satoru; Ohdo, Shigehiro; Kubota, Toshiaki

    2015-11-06

    Clock gene regulates the circadian rhythm of various physiological functions. The expression of clock gene has been shown to be attenuated by certain drugs, resulting in a rhythm disorder. Mitomycin C (MMC) is often used in combination with ophthalmic surgery, especially in trabeculectomy, a glaucoma surgical procedure. The purpose of this study was to investigate the influence of MMC on clock gene expression in fibroblasts, the target cells of MMC. Following MMC treatment, Bmal1 mRNA levels was significantly decreased, whereas Dbp, Per1, and Rev-erbα mRNA levels were significantly increased in the mouse fibroblast cell line NIH3T3 cells. Microarray analysis was performed to explore of the gene(s) responsible for MMC-induced alteration of clock gene expression, and identified Nr3c1 gene encoding glucocorticoid receptor (GR) as a candidate. MMC suppressed the induction of Per1 mRNA by dexamethasone (DEX), ligand of GR, in NIH3T3 cells. MMC also modulated the DEX-driven circadian oscillations of Per2::Luciferase bioluminescence in mouse-derived ocular fibroblasts. Our results demonstrate a previously unknown effect of MMC in GR signaling and the circadian clock system. The present findings suggest that MMC combined with trabeculectomy could increase the risk for a local circadian rhythm-disorder at the ocular surface.

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

    PubMed

    Kowalska, Katarzyna; Olejnik, Anna; Rychlik, Joanna; Grajek, Włodzimierz

    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 PPARγ, C/EBPα 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.

  18. Increased association of dynamin II with myosin II in ras transformed NIH3T3 cells.

    PubMed

    Jeong, Soon-Jeong; Kim, Su-Gwan; Yoo, Jiyun; Han, Mi-Young; Park, Joo-Cheol; Kim, Heung-Joong; Kang, Seong-Soo; Choi, Baik-Dong; Jeong, Moon-Jin

    2006-08-01

    Dynamin has been implicated in the formation of nascent vesicles through both endocytic and secretory pathways. However, dynamin has recently been implicated in altering the cell membrane shape during cell migration associated with cytoskeleton-related proteins. Myosin II has been implicated in maintaining cell morphology and in cellular movement. Therefore, reciprocal immunoprecipitation was carried out to identify the potential relationship between dynamin II and myosin II. The dynamin II expression level was higher when co-expressed with myosin II in Ras transformed NIH3T3 cells than in normal NIH3T3 cells. Confocal microscopy also confirmed the interaction between these two proteins. Interestingly, exposing the NIH3T3 cells to platelet-derived growth factor altered the interaction and localization of these two proteins. The platelet-derived growth factor treatment induced lamellipodia and cell migration, and dynamin II interacted with myosin II. Grb2, a 24 kDa adaptor protein and an essential element of the Ras signaling pathway, was found to be associated with dynamin II and myosin II gene expression in the Ras transformed NIH3T3 cells. These results suggest that dynamin II acts as an intermediate messenger in the Ras signal transduction pathway leading to membrane ruffling and cell migration.

  19. Tubby-like protein superfamily member PLSCR3 functions as a negative regulator of adipogenesis in mouse 3T3-L1 preadipocytes by suppressing induction of late differentiation stage transcription factors

    PubMed Central

    Inokawa, Akira; Inuzuka, Tatsutoshi; Takahara, Terunao; Shibata, Hideki; Maki, Masatoshi

    2015-01-01

    PLSCR3 (phospholipid scramblase 3, Scr3) belongs to the superfamily of membrane-associated transcription regulators named Tubby-like proteins (TULPs). Physiological phospholipid scrambling activities of PLSCRs in vivo have been skeptically argued, and knowledge of the biological functions of Scr3 is limited. We investigated the expression of Scr3 during differentiation of mouse 3T3-L1 preadipocytes by Western blotting (WB) and by reverse-transcription and real-time quantitative PCR (RT-qPCR). The Scr3 protein decreased during 3T3-L1 differentiation accompanied by a reduction in the mRNA level, and there was a significant increase in the amount of Scr3 protein secreted into the culture medium in the form of extracellular microvesicles (exosomes). On the other hand, Scr3 expression did not significantly decrease, and the secretion of Scr3 in 3T3 Swiss-albino fibroblasts (a parental cell-line of 3T3-L1) was not increased by differentiation treatment. Overexpression of human Scr3 during 3T3-L1 differentiation suppressed triacylglycerol accumulation and inhibited induction of the mRNAs of late stage pro-adipogenic transcription factors [CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ)] and X-box-binding protein 1 (XBP1). Expression of early stage pro-adipogenic transcription factors (C/EBPβ and C/EBPδ) was not significantly affected. These results suggest that Scr3 functions as a negative regulator of adipogenesis in 3T3-L1 cells at a specific differentiation stage and that decrease in the intracellular amount of Scr3 protein caused by reduction in Scr3 mRNA expression and enhanced secretion of Scr3 protein appears to be important for appropriate adipocyte differentiation. PMID:26677203

  20. Vaspin promotes 3T3-L1 preadipocyte differentiation

    PubMed Central

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

    2015-01-01

    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 γ 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 γ, C/EBPα, 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/EBPβ, peroxisome proliferator-activated γ, C/EBPα, 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

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

    PubMed Central

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

    2013-01-01

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

  2. Berberine activates GLUT1-mediated glucose uptake in 3T3-L1 adipocytes.

    PubMed

    Kim, So Hui; Shin, Eun-Jung; Kim, Eun-Do; Bayaraa, Tsenguun; Frost, Susan Cooke; Hyun, Chang-Kee

    2007-11-01

    It has recently been known that berberine, an alkaloid of medicinal plants, has anti-hyperglycemic effects. To explore the mechanism underlying this effect, we used 3T3-L1 adipocytes for analyzing the signaling pathways that contribute to glucose transport. Treatment of berberine to 3T3-L1 adipocytes for 6 h enhanced basal glucose uptake both in normal and in insulin-resistant state, but the insulin-stimulated glucose uptake was not augmented significantly. Inhibition of phosphatidylinositol 3-kinase (PI 3-K) by wortmannin did not affect the berberine effect on basal glucose uptake. Berberine did not augment tyrosine phosphorylation of insulin receptor (IR) and insulin receptor substrate (IRS)-1. Further, berberine had no effect on the activity of the insulin-sensitive downstream kinase, atypical protein kinase C (PKCzeta/lambda). However, interestingly, extracellular signal-regulated kinases (ERKs), which have been known to be responsible for the expression of glucose transporter (GLUT)1, were significantly activated in berberine-treated 3T3-L1 cells. As expected, the level of GLUT1 protein was increased both in normal and insulin-resistant cells in response to berberine. But berberine affected the expression of GLUT4 neither in normal nor in insulin-resistant cells. In addition, berberine treatment increased AMP-activated protein kinase (AMPK) activity in 3T3-L1 cells, which has been reported to be associated with GLUT1-mediated glucose uptake. Together, we concluded that berberine increases glucose transport activity of 3T3-L1 adipocytes by enhancing GLUT1 expression and also stimulates the GLUT1-mediated glucose uptake by activating GLUT1, a result of AMPK stimulation.

  3. Cirsium japonicum flavones enhance adipocyte differentiation and glucose uptake in 3T3-L1 cells.

    PubMed

    Liao, Zhiyong; Wu, Zhihua; Wu, Mingjiang

    2012-01-01

    Cirsium japonicum flavones have been demonstrated to possess anti-diabetic effects in diabetic rats, but the functional mechanism remains unknown. The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) plays an important role in glucose and lipid homeostasis. In this study, we report the effects of Cirsium japonicum flavones (pectolinarin and 5,7-dihydroxy-6,4-dimethoxy flavone) on PPARγ activation, adipocyte differentiation, and glucose uptake in 3T3-L1 cells. Reporter gene assays and Oil Red O staining showed that Cirsium japonicum flavones induced PPARγ activation and enhanced adipocyte differentiation of 3T3-L1 cells in a dose-dependent manner. In addition, Cirsium japonicum flavones increased the expression of PPARγ target genes, such as adiponectin and glucose transporter 4 (GLUT4), and enhanced the translocation of intracellular GLUT4 to the plasma membrane. In mature 3T3-L1 adipocytes, Cirsium japonicum flavones significantly enhanced the basal and insulin-stimulated glucose uptake. The flavones-induced effects in 3T3-L1 cells were abolished by the PPARγ antagonist, GW9662, and by the phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin. This study suggests that Cirsium japonicum flavones promote adipocyte differentiation and glucose uptake by inducing PPARγ activation and then modulating the insulin signaling pathway in some way, which could benefit diabetes patients.

  4. 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.

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

    SciTech Connect

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

    1986-02-01

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

  6. Cytokine-mediated PGE2 expression in human colonic fibroblasts.

    PubMed

    Kim, E C; Zhu, Y; Andersen, V; Sciaky, D; Cao, H J; Meekins, H; Smith, T J; Lance, P

    1998-10-01

    We investigated prostanoid biogenesis in human colonic fibroblasts (CCD-18Co and 5 primary fibroblast cultures) and epithelial cell lines (NCM460, T84, HT-29, and LS 174T) and the effect of PGE2 on fibroblast morphology. Cytokine-stimulated PGE2 production was measured. PGH synthase-1 and -2 (PGHS-1 and -2) protein and mRNA expression were evaluated. Basal PGE2 levels were low in all cell types (0.15-6.47 ng/mg protein). Treatment for 24 h with interleukin-1beta (IL-1beta; 10 ng/ml) or tumor necrosis factor-alpha (50 ng/ml), respectively, elicited maximal 25- and 6-fold inductions of PGE2 synthesis in CCD-18Co cultures and similar results in primary fibroblast cultures; maximal inductions with IL-1beta in colonic epithelial cell lines were from zero to fivefold. Treatment of CCD-18Co fibroblasts with IL-1beta caused maximal 21- and 53-fold increases, respectively, in PGHS-2 protein and mRNA levels without altering PGHS-1 expression. PGE2 (0.1 micromol/l) elicited a dramatic shape change in selected fibroblasts. Colonic fibroblasts are potentially important as cytokine targets and a source of and target for colonic prostanoids in vivo.

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

    PubMed

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

    2010-11-01

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

  8. CP27 affects viability, proliferation, attachment and gene expression in embryonic fibroblasts.

    PubMed

    Luan, X; Diekwisch, T G H

    2002-08-01

    CP27 is a gene that has been cloned from an E11 early embryonic library and has been suggested to mediate early organogenesis (Diekwisch et al., 1999, Gene 235, 19). We have hypothesized that CP27 exhibits its effects on organogenesis by affecting individual cell function. Based on the CP27 expression pattern we have selected the CP27 expressing embryonic fibroblast cell line BALB/c 3T3 to determine the effects of CP27 on cell function. CP27 loss of function strategies were performed by adding 5, 12.5 or 25 micro g/ml anti-CP27 antibody to cultured BALB/c 3T3 cells and comparing the results to controls in which identical concentrations of rabbit serum were added to the culture medium. Other controls included an antibody against another extracellular matrix protein amelogenin (negative control) and anti-CP27 antibodies directed against other areas of the CP27 molecule (positive control). Following cell culture, cell viability, apoptosis, cell proliferation, cell shape, cellular attachment and fibronectin matrix production were assayed using MTT colourimetric assay, BrdU staining, morphometry, immunostaining and western blot analysis. Block of CP27 function using an antibody strategy resulted in the following significant changes: (i) reduced viability, (ii) increased number of apoptotic cells, (iii) reduced proliferation, (iv) alterations in cell shape, (v) loss of attachment, and (vi) reduction in fibronectin matrix production. There was also a redistribution in fibronectin matrix organization demonstrated by immunohistochemistry. We conclude that CP27 plays an important role in the maintance of normal cell function and that CP27 block leads to significant changes in cellular behaviour.

  9. The 3T3-L1 adipocyte glycogen proteome

    PubMed Central

    2013-01-01

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

  10. 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

  11. 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

  12. 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.

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

    PubMed Central

    Yang, Soo Jin; Park, Na-Young

    2014-01-01

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

  14. 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 γH2AX 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 < 0.05) followed by a slight decrease when the exposure continued for as long as 8 h. No significant effect on the number of γH2AX was detected after EMR exposure. The percentage of late-apoptotic cells in the EMR-exposed group was significantly higher than that in the sham-exposed groups (p < 0.05). These results indicate that an 1800-MHz EMR enhances ROS formation and promotes apoptosis in NIH/3T3 cells.

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

    SciTech Connect

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

    1989-05-01

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

  16. 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-α), and peroxisome proliferator-activated receptor gamma (PPAR-γ). 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

  17. WEHI-3 cells inhibit adipocyte differentiation in 3T3-L1 cells

    SciTech Connect

    Lai, Jing; Liu, Gexiu; Yan, Guoyao; He, Dongmei; Zhou, Ying; Chen, Shengting

    2015-06-26

    By investigating the anti-adipogenic effects of WEHI-3 cells – a murine acute myelomonocytic leukemia cell line – we sought to improve the efficiency of hematopoietic stem cell transplantation (HSCT). Analysis of Oil Red O staining and the expression of adipogenic genes, including PPARγ, C/EBPα, FAS and LPL, indicated that WEHI-3 cells significantly inhibited 3T3-L1 mouse preadipocyte cells from differentiating into adipocytes. In vivo, fat vacuoles in mice injected with WEHI-3 cells were also remarkably reduced in the murine bone marrow pimelosis model. Moreover, the key gene in the Rho signaling pathway, ROCKII, and the key gene in the Wnt signaling pathway, β-catenin, were both upregulated compared with the control group. siRNA-mediated knockdown of ROCKII and β-catenin reversed these WEHI-3-mediated anti-adipogenic effects. Taken together, these data suggest that WEHI-3 cells exert anti-adipogenic effects and that both ROCKII and β-catenin are involved in this process. - Highlights: • WEHI-3, an acute myelomonocytic leukemia cell line, inhibited 3T3-L1 preadipocyte from differentiating into adipocyte. • WEHI-3 cells can arrest 3T3-L1 cells in G0/G1 phase by secreting soluble factors and thus inhibit their proliferation. • WEHI-3 cells reduced bone marrow pimelosis in the murine model. • Both ROCKII and β-catenin were involved in the WEHI-3-mediated anti-adipogenic effects.

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

    SciTech Connect

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

    1987-09-01

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

  19. Increased expression of fibroblast growth factors in a rabbit skeletal muscle model of exercise conditioning.

    PubMed Central

    Morrow, N G; Kraus, W E; Moore, J W; Williams, R S; Swain, J L

    1990-01-01

    Increased tonic contractile activity from exercise or electrical stimulation induces a variety of changes in skeletal muscle, including vascular growth, myoblast proliferation, and fast to slow fiber type conversion. Little is known about the cellular control of such changes, but pleiotropic biochemical modulators such as fibroblast growth factors (FGFs) may be involved in this response and thus may be regulated in response to such stimuli. We examined the regulation of FGF expression in an in vivo model of exercise conditioning previously shown to exhibit vascular growth and fast to slow fiber conversion. FGFs were extracted by heparin-affinity chromatography from extensor digitorum longus muscles of adult rabbits subjected to chronic motor nerve stimulation at 10 Hz. Growth factor activity (expressed in growth factor units [GFUs]) of muscle stimulated for 3 and 21 d was assayed by [3H]thymidine incorporation in 3T3 fibroblasts and compared with that present in the contralateral unstimulated muscle. A small increase in heparin-binding mitogenic activity was observed as early as 3 d of stimulation, and by 21 d mitogenic activity increased significantly when normalized to either wet weight (stimulated, 287 +/- 61 GFU/g; unstimulated, 145 +/- 39 GFU/g) or to protein (stimulated, 5.3 +/- 1.1 GFU/mg; unstimulated, 2.2 +/- 0.6 GFU/mg) (+/- SE, P less than 0.05). Western analysis demonstrated increased amounts of peptides with immunological identity to acidic and basic FGFs in stimulated muscle. The increase in FGF content observed in this study is synchronous with neovascularization, myoblast proliferation, and fast to slow fiber type conversion previously shown in this model. These results demonstrate that increased expression of FGFs is associated with motor nerve stimulation and increased tonic contractile activity of skeletal muscle, and suggests that these proteins may play a regulatory role in the cellular changes that occur during exercise conditioning. Images

  20. Combination therapy with catechins and caffeine inhibits fat accumulation in 3T3-L1 cells

    PubMed Central

    Zhu, Xiaojuan; Yang, Licong; Xu, Feng; Lin, Lezhen; Zheng, Guodong

    2017-01-01

    Catechins and caffeine, which are green tea components, have a slimming effect; however, the combinational effect of fat metabolism in 3T3-L1 cells remains unclear. In the present study, 3T3-L1 cells were treated with catechins and caffeine in combination, and it was found that combination therapy with catechins and caffeine markedly reduced intracellular fat accumulation, mRNA expression levels of peroxisome proliferator-activated receptor-γ and CCAAT/enhancer-binding protein α in the early stage of cell differentiation were significantly reduced, and mRNA expression of fatty acid synthetase(FAS) andglycerol-3-phosphate dehydrogenase protein expression levels of FAS were downregulated. Noradrenaline-induced lipolysis was enhanced by caffeine, which markedly increased the protein expression of adipose triglyceride lipase and hormone sensitive lipase. These results indicated that combination therapy with catechins and caffeine synergistically inhibited lipid accumulation by regulating the gene and protein expression levels of lipid metabolism-related enzymes. Therefore, catechins and caffeine combination therapy has potential as a functional food that may be used to prevent obesity and lifestyle-associated diseases. PMID:28352352

  1. 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

  2. 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.

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

    PubMed

    Smith, M R; Ryu, S H; Suh, P G; Rhee, S G; Kung, H F

    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 (100-700 micrograms/ml) into quiescent NIH 3T3 cells, a time- and dose-dependent induction of DNA synthesis occurred, as demonstrated by [3H]thymidine incorporation into nuclear DNA. In addition, approximately to 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 (1 mg/ml) 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 (approximately 10,000 molecules per cell) to override the cellular G0 block, inducing DNA synthesis and morphologic transformation of growth-arrested fibroblast cells.

  4. Expression of the endocannabinoid system in fibroblasts and myofascial tissues.

    PubMed

    McPartland, John M

    2008-04-01

    The endocannabinoid (eCB) system, like the better-known endorphin system, consists of cell membrane receptors, endogenous ligands and ligand-metabolizing enzymes. Two cannabinoid receptors are known: CB(1) is principally located in the nervous system, whereas CB(2) is primarily associated with the immune system. Two eCB ligands, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are mimicked by cannabis plant compounds. The first purpose of this paper was to review the eCB system in detail, highlighting aspects of interest to bodyworkers, especially eCB modulation of pain and inflammation. Evidence suggests the eCB system may help resolve myofascial trigger points and relieve symptoms of fibromyalgia. However, expression of the eCB system in myofascial tissues has not been established. The second purpose of this paper was to investigate the eCB system in fibroblasts and other fascia-related cells. The investigation used a bioinformatics approach, obtaining microarray data via the GEO database (www.ncbi.nlm.nih.gov/geo/). GEO data mining revealed that fibroblasts, myofibroblasts, chondrocytes and synoviocytes expressed CB(1), CB(2) and eCB ligand-metabolizing enzymes. Fibroblast CB(1) levels nearly equalled levels expressed by adipocytes. CB(1) levels upregulated after exposure to inflammatory cytokines and equiaxial stretching of fibroblasts. The eCB system affects fibroblast remodeling through lipid rafts associated with focal adhesions and dampens cartilage destruction by decreasing fibroblast-secreted metalloproteinase enzymes. In conclusion, the eCB system helps shape biodynamic embryological development, diminishes nociception and pain, reduces inflammation in myofascial tissues and plays a role in fascial reorganization. Practitioners wield several tools that upregulate eCB activity, including myofascial manipulation, diet and lifestyle modifications, and pharmaceutical approaches.

  5. 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 β3-adrenergic receptor (AR). •Nebivolol stimulates mitochondrial DNA replication and protein expression. •Nebivolol promotes mitochondrial synthesis via activation of eNOS by β3-AR. -- Abstract: Nebivolol is a third-generation β-adrenergic receptor (β-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-γ coactivator-1α (PGC-1α), 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 β3-AR blocker SR59230A markedly attenuated PGC-1α, 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 β3-AR receptors.

  6. Gsalpha signalling suppresses PPARgamma2 generation and inhibits 3T3L1 adipogenesis.

    PubMed

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

    2009-08-01

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

  7. Tropoelastin regulates chemokine expression in fibroblasts in Costello syndrome

    SciTech Connect

    Tatano, Yutaka; Fujinawa, Reiko; Kozutsumi, Yasunori; Takahashi, Tsutomu; Tsuji, Daisuke; Takeuchi, Naohiro; Tsuta, Kohji; Takada, Goro; Sakuraba, Hitoshi; Itoh, Kohji

    2008-08-08

    Costello syndrome is a multiple congenital anomaly associated with growth and mental retardation, cardiac and skeletal anomalies, and a predisposition to develop neoplasia. Comprehensive expression analysis revealed remarkable up-regulation of several cytokines and chemokines including Gro family proteins, interleukin-1{beta} (IL-1{beta}), IL-8 and MCP-1 but down-regulation of extracellular matrix components including collagens and proteoglycans of skin fibroblasts derived from a Japanese Costello syndrome patient characterized by significantly reduced tropoelastin mRNA, impaired elastogenesis and enhanced cell proliferation. In contrast, decreases in these chemokines and IL-1{beta} expression were observed in Costello fibroblastic cell lines stably expressing the bovine tropoelastin (btEln) gene and in restored elastic fibers. These results strongly suggest that the human TE gene (ELN) transfer could be applicable for the gene therapy of a group of Costello syndrome patients with reduced ELN gene expression.

  8. Tocotrienol suppresses adipocyte differentiation and Akt phosphorylation in 3T3-L1 preadipocytes.

    PubMed

    Uto-Kondo, Harumi; Ohmori, Reiko; Kiyose, Chikako; Kishimoto, Yoshimi; Saito, Hisako; Igarashi, Osamu; Kondo, Kazuo

    2009-01-01

    In vivo studies show that alpha-tocotrienol and gamma-tocotrienol accumulate in adipose tissue. Furthermore, a recent study reports that the oral administration of gamma-tocotrienol from a tocotrienol-rich fraction from palm oil (TRF) decreases body fat levels in rats. The objective of this study was to evaluate the effect of TRF and its components on adipocyte differentiation in 3T3-L1 preadipocytes, which differentiated into adipocytes in the presence of 1.8 micromol/L insulin. TRF suppressed the insulin-induced mRNA expression of adipocyte-specific genes such as PPARgamma, adipocyte fatty acid-binding protein (aP2), and CCAAT/enhancer-binding protein-alpha (C/EBPalpha) compared with the differentiation of 3T3-L1 preadipocytes into adipocytes only in the presence of insulin. To confirm the suppressive effect of TRF, the major components of TRF, such as alpha-tocotrienol, gamma-tocotrienol, and alpha-tocopherol, were investigated. Alpha-tocotrienol and gamma-tocotrienol decreased the insulin-induced PPARgamma mRNA expression by 55 and 90%, respectively, compared with insulin, whereas alpha-tocopherol increased the mRNA expression. In addition, gamma-tocotrienol suppressed the insulin-induced aP2 and C/EBPalpha mRNA expression, triglyceride accumulation, and PPARgamma protein levels compared with insulin. The current results also revealed that gamma-tocotrienol inhibited the insulin-stimulated phosphorylation of Akt but not extracellular signal-regulated kinase (ERK)1/2 in the insulin signaling pathway of 3T3-L1 preadipocytes. Thus, the antiadipogenic effect of TRF depends on alpha-tocotrienol and gamma-tocotrienol, and gamma-tocotrienol may be a more potent inhibitor of adipogenesis than alpha-tocotrienol. Therefore, the results of this study suggest that tocotrienol suppresses insulin-induced differentiation and Akt phosphorylation in 3T3-L1 preadipocytes. Furthermore, tocotrienol could act as an antiadipogenic vitamin in the nutrient-mediated regulation of body

  9. Calcium pantothenate modulates gene expression in proliferating human dermal fibroblasts.

    PubMed

    Wiederholt, Tonio; Heise, Ruth; Skazik, Claudia; Marquardt, Yvonne; Joussen, Sylvia; Erdmann, Kati; Schröder, Henning; Merk, Hans F; Baron, Jens Malte

    2009-11-01

    Topical application of pantothenate is widely used in clinical practice for wound healing. Previous studies identified a positive effect of pantothenate on migration and proliferation of cultured fibroblasts. However, these studies were mainly descriptive with no molecular data supporting a possible model of its action. In this study, we first established conditions for an in vitro model of pantothenate wound healing and then analysed the molecular effects of pantothenate. To test the functional effect of pantothenate on dermal fibroblasts, cells were cultured and in vitro proliferation tests were performed using a standardized scratch test procedure. For all three donors analysed, a strong stimulatory effect of pantothenate at a concentration of 20 microg/ml on the proliferation of cultivated dermal fibroblasts was observed. To study the molecular mechanisms resulting in the proliferative effect of pantothenate, gene expression was analysed in dermal fibroblasts cultivated with 20 microg/ml of pantothenate compared with untreated cells using the GeneChip Human Exon 1.0 ST Array. A number of significantly regulated genes were identified including genes coding for interleukin (IL)-6, IL-8, Id1, HMOX-1, HspB7, CYP1B1 and MARCH-II. Regulation of these genes was subsequently verified by quantitative real-time polymerase chain reaction analysis. Induction of HMOX-1 expression by pantothenol and pantothenic acid in dermal cells was confirmed on the protein level using immunoblots. Functional studies revealed the enhanced suppression of free radical formation in skin fibroblasts cultured with panthenol. In conclusion, these studies provided new insight in the molecular mechanisms linked to the stimulatory effect of pantothenate and panthenol on the proliferation of dermal fibroblasts.

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

    PubMed

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

    2007-12-01

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

  11. Behavior of a fluorescent analogue of calmodulin in living 3T3 cells

    PubMed Central

    1985-01-01

    We have prepared and partially characterized a lissamine-rhodamine B fluorescent analogue of calmodulin, LRB-CM. The analogue had a dye/protein ratio of approximately 1.0 and contained no free dye or contaminating labeled proteins. LRB-CM was indistinguishable from native calmodulin upon SDS PAGE and in assays of phosphodiesterase and myosin light chain kinase. The emission spectrum of LRB-CM was insensitive to changes in pH, ionic strength, and temperature over the physiological range, but the apparent quantum yield was influenced somewhat by divalent cation concentration. LRB-CM injected into living Swiss 3T3 fibroblasts became associated with nitrobenzoxadiazole- phallacidin staining stress fibers in some interphase cells. LRB-CM and acetamidofluorescein-labeled actin co-injected into the same cell both became associated with fibers in some cells, but in most cases association of the two analogues with fibers was mutually exclusive. This suggests that calmodulin may differ from actin in the timing of incorporation into stress fibers or that we have distinguished distinct populations of stress fibers. We were able to detect no direct interaction of LRB-CM with actin by fluorescence photobleaching recovery (FRAP) of aqueous solutions. Interaction of LRB-CM with myosin light chain kinase also was not detected by FRAP. This suggests that the mean lifetime of the calmodulin-myosin light chain kinase complex is too short to affect the diffusion coefficient of calmodulin. We examined various fluorescent derivatives of proteins and dextrans as suitable control molecules for quantitative fluorescent analogue cytochemistry in living cells. Fluorescein isothiocyanate-dextrans were found to be preferable to all the proteins tested, since their mobilities in cytoplasm were inversely dependent on molecular size and there was no evidence of binding to intracellular components. In contrast, FRAP of LRB-CM in the cytoplasm of living 3T3 cells suggested that the analogue

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

    PubMed Central

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

    1994-01-01

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

  13. 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.

  14. 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-α (TNF-α), monocyte chemotactic protein-1 (MCP-1), and resistin mRNA expression, similarly to treatment with troglitazone, which is a PPARγ 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

  15. Single Synonymous Mutations in KRAS Cause Transformed Phenotypes in NIH3T3 Cells

    PubMed Central

    Waters, Andrew M.; Bagni, Rachel; Portugal, Franklin; Hartley, James L.

    2016-01-01

    Synonymous mutations in the KRAS gene are clustered at G12, G13, and G60 in human cancers. We constructed 9 stable NIH3T3 cell lines expressing KRAS, each with one of these synonymous mutations. Compared to the negative control cell line expressing the wild type human KRAS gene, all the synonymous mutant lines expressed more KRAS protein, grew more rapidly and to higher densities, and were more invasive in multiple assays. Three of the cell lines showed dramatic loss of contact inhibition, were more refractile under phase contrast, and their refractility was greatly reduced by treatment with trametinib. Codon usage at these glycines is highly conserved in KRAS compared to HRAS, indicating selective pressure. These transformed phenotypes suggest that synonymous mutations found in driver genes such as KRAS may play a role in human cancers. PMID:27684555

  16. Isorhamnetin represses adipogenesis in 3T3-L1 cells.

    PubMed

    Lee, Jongsung; Jung, Eunsun; Lee, Jienny; Kim, Saebom; Huh, Sungran; Kim, Youngsoo; Kim, Yongwoo; Byun, Sang Yo; Kim, Yeong-Shik; Park, Deokhoon

    2009-02-01

    Adipocyte dysfunction is strongly associated with the development of obesity, which is a major risk factor for many disorders including diabetes, hypertension, and heart disease. It is generally accepted that the regulation of adipogenesis or adipokines expression prevents obesity. In this study, we show that isorhamnetin inhibits adipocyte differentiation, as evidenced by reduced triglyceride (TG) accumulation and glycerol-3-phosphate dehydrogenase (GPDH) activity. At the molecular level, the mRNA expression levels of peroxidase proliferator-activated receptor-gamma (PPAR-gamma) and CCAAT/enhancer-binding protein-alpha (C/EBP-alpha), which are the major adipogenic transcription factors, were markedly reduced by isorhamnetin. However, the mRNA levels of C/EBP-beta and -delta, the upstream regulators of PPAR-gamma and C/EBP-alpha, were not reduced by isorhamnetin. Moreover, the mRNA levels of PPAR-gamma target genes such as lipoprotein lipase (LPL), CD36, aP2, and liver X receptor-alpha (LXR-alpha) were downregulated by isorhamnetin. We also showed that isorhamnetin inhibits the expression and secretion of adiponectin, and the results of adiponectin promoter assays suggest the inhibition of PPAR-gamma expression as a possible mechanism underlying the isorhamnetin-mediated effects. Taken together, these results indicate that isorhamnetin inhibits adipogenesis through downregulation of PPAR-gamma and C/EBP-alpha.

  17. 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 PPARγ 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

  18. A Marfan syndrome gene expression phenotype in cultured skin fibroblasts

    PubMed Central

    Yao, Zizhen; Jaeger, Jochen C; Ruzzo, Walter L; Morale, Cecile Z; Emond, Mary; Francke, Uta; Milewicz, Dianna M; Schwartz, Stephen M; Mulvihill, Eileen R

    2007-01-01

    Background Marfan syndrome (MFS) is a heritable connective tissue disorder caused by mutations in the fibrillin-1 gene. This syndrome constitutes a significant identifiable subtype of aortic aneurysmal disease, accounting for over 5% of ascending and thoracic aortic aneurysms. Results We used spotted membrane DNA macroarrays to identify genes whose altered expression levels may contribute to the phenotype of the disease. Our analysis of 4132 genes identified a subset with significant expression differences between skin fibroblast cultures from unaffected controls versus cultures from affected individuals with known fibrillin-1 mutations. Subsequently, 10 genes were chosen for validation by quantitative RT-PCR. Conclusion Differential expression of many of the validated genes was associated with MFS samples when an additional group of unaffected and MFS affected subjects were analyzed (p-value < 3 × 10-6 under the null hypothesis that expression levels in cultured fibroblasts are unaffected by MFS status). An unexpected observation was the range of individual gene expression. In unaffected control subjects, expression ranges exceeding 10 fold were seen in many of the genes selected for qRT-PCR validation. The variation in expression in the MFS affected subjects was even greater. PMID:17850668

  19. Piromelatine decreases triglyceride accumulation in insulin resistant 3T3-L1 adipocytes: role of ATGL and HSL.

    PubMed

    Wang, Ping-Ping; She, Mei-Hua; He, Ping-Ping; Chen, Wu-Jun; Laudon, Moshe; Xu, Xuan-Xuan; Yin, Wei-Dong

    2013-08-01

    Piromelatine, a novel investigational multimodal sleep medicine, is developed for the treatment of patients with primary and co-morbid insomnia. Piromelatine has been shown to inhibit weight gain and improve insulin sensitivity in high-fat/high-sucrose-fed (HFHS) rats. Considering that piromelatine has also been implicated in lowering of triglyceride levels in HFHS rats, this work elucidated whether this effect involves in the regulation of adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) in triglyceride (TG) metabolism. In this study, we investigated the effects of piromelatine and MT2 receptors inhibition on TG content, insulin-stimulated glucose uptake, and the expressions of ATGL and HSL in 3T3-L1 adipocytes preincubated in high glucose and high insulin (HGI) conditions. Our results showed that culturing 3T3-L1 adipocytes under HGI conditions increased triglyceride accumulation with concomitant decrease of ATGL and HSL expression, inducing insulin resistance in 3T3-L1 adipocytes. We also found that triglyceride accumulation was significantly inhibited and the levels of ATGL/HSL increased after melatonin or piromelatine treatment. The effects of melatonin/piromelatine (10 nM) were counteracted by pretreatment with the relatively selective MT2 receptor antagonist luzindole (100 nM). In this study, our data demonstrate that piromelatine reverses high glucose and high insulin-induced triglyceride accumulation in 3T3-L1 adipocytes, possibly through up-regulating of ATGL and HSL expression via a melatonin-dependent manner.

  20. Berberine inhibits 3T3-L1 adipocyte differentiation through the PPARgamma pathway.

    PubMed

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

    2006-09-22

    Berberine (BBR), a compound purified from Cortidis rhizoma, reduces serum cholesterol, triglycerides, and LDL-cholesterol of hypercholesterolemic patients and high fat diet fed animals, and increases hepatic LDLR mRNA and protein levels through a post-transcriptional mechanism. BBR also enhances the hypoglycemic action of insulin in diabetic animal models. Here, we show that BBR inhibits the differentiation of 3T3-L1 preadipocytes induced by DM and suppresses the mitotic clonal expansion of 3T3-L1 preadipocytes in a time- and dose-dependent manner. Gene expression analysis and Western blot analysis reveal that the BBR inhibits the mRNA and protein levels of adipogenesis related transcription factors PPARgamma and C/EBPalpha and their upstream regulator, C/EBPbeta. Reporter gene assays demonstrate that the full-length PPARgamma and alpha transcription activities are inhibited by BBR. Using real-time PCR, we have also found that the PPAR target genes that are involved in adipocyte differentiation, such as aP2, CD36, ACO, LPL, and other adipocyte markers, are suppressed by BBR. These studies suggest that BBR works on multiple molecular targets as an inhibitor of PPARgamma and alpha, and is a potential weight reducing, hypolipidemic, and hypoglycemic drug.

  1. Effects of Berberine on Adipose Tissues and Kidney Function in 3T3-L1 Cells and Spontaneously Hypertensive Rats.

    PubMed

    Kishimoto, Aya; Dong, Shi-Fen; Negishi, Hiroko; Yasui, Naomi; Sun, Jian-Ning; Ikeda, Katsumi

    2015-09-01

    We aimed to investigate the effect of berberine on adipose tissues, as well as its effect on renal injury in 3T3-L1 cells and spontaneously hypertensive rats. 3T3-L1 cells were cultured and treated with berberine (5-20 pM) from days 3 to 8. Berberine added to the cultured medium could significantly down-regulate transcription factors, including CCAAT/enhancer binding protein β, CCAAT/enhancer binding protein a, and peroxisome pro liferator-activated receptor y, and suppress peroxisome proliferator-activated receptor target genes, such as adipocyte fatty acid binding protein and fatty acid synthase, and inhibit 3T3-Ll fibroblast differentiation to adipocytes. Male spontaneously hypertensive rats received either 150 mg/day of berberine or saline orally for 8 weeks. Compared with the control, berberine-treated rats exhibited significant reductions in body weight gain (p < 0.05), as well as retroperitoneal and mesenteric adipose tissues (p < 0.05). Berberine-treated rats significantly decreased urinary albumin excretion, a marker of renal injury (p < 0.05). Long-term treatment with berberine decreased the adipose tissues weight and attenuated renal injury in spontaneously hypertensive rats. Based on these results, berberine has an important role in regulating adipose tissues. These results suggest the protective effect of berberine on metabolic syndrome related diseases, such as renal injury.

  2. The role and possible mechanism of lncRNA U90926 in modulating 3T3-L1 preadipocyte differentiation

    PubMed Central

    Chen, J; Liu, Y; Lu, S; Yin, L; Zong, C; Cui, S; Qin, D; Yang, Y; Guan, Q; Li, X; Wang, X

    2017-01-01

    Background: Obesity is a risk factor for metabolic diseases, while preadipocyte differentiation or adipogenesis is closely related to obesity occurrence. Long noncoding RNAs (lncRNAs) are a unique class of transcripts in regulation of a variety of biological processes. Using cDNA microarray, we found lncRNA U90926 is negatively correlated with 3T3-L1 preadipocyte differentiation. Objective: The aim of this study was to explore the role of lncRNA U90926 (lnc-U90926) in adipogenesis and the underlying mechanisms. Methods: Quantitative real-time PCR (qPCR) was performed to determine lnc-U90926 expression in 3T3-L1 preadipocytes, differentiated adipocytes, and in adipose tissues form mice. RNA fluorescent in situ hybridization (FISH) was performed to determine the localization of lnc-U90926 in 3T3-L1 preadipocytes. The effects of lnc-U90926 on 3T3-L1 adipogenesis were analyzed with lentivirus-mediated gain- and loss-of-function experiments. Lipid accumulation was evaluated by oil red O staining; several adipogenesis makers were analyzed by qPCR and western blotting. Dual luciferase assay was applied to explore the transactivation of target genes modulated by lnc-U90926. All measurements were performed at least for three times. Results: Lnc-U90926 expression decreased along the differentiation of 3T3-L1 preadipocytes. In mice, lnc-U90926 is predominantly expressed in adipose tissue. Obese mice have lower lnc-U90926 expression in subcutaneous and visceral adipose tissue than non-obese mice. FISH results showed that lnc-U90926 was mainly located in the cytoplasm. Overexpression lnc-U90926 attenuated 3T3-L1 adipocyte differentiation as evidenced by its ability to inhibit lipid accumulation, to decrease the mRNA levels of peroxisome proliferator-activated receptor gamma 2 (PPARγ2), fatty acid binding protein 4 (FABP4) and adiponectin (AdipoQ) as well as to reduce the protein levels of PPARγ and FABP4 (P<0.05). Knockdown of lnc-U90926 showed opposite effects, which

  3. Adhesion, Proliferation and Migration of NIH/3T3 Cells on Modified Polyaniline Surfaces

    PubMed Central

    Rejmontová, Petra; Capáková, Zdenka; Mikušová, Nikola; Maráková, Nela; Kašpárková, Věra; Lehocký, Marián; Humpolíček, Petr

    2016-01-01

    Polyaniline shows great potential and promises wide application in the biomedical field thanks to its intrinsic conductivity and material properties, which closely resemble natural tissues. Surface properties are crucial, as these predetermine any interaction with biological fluids, proteins and cells. An advantage of polyaniline is the simple modification of its surface, e.g., by using various dopant acids. An investigation was made into the adhesion, proliferation and migration of mouse embryonic fibroblasts on pristine polyaniline films and films doped with sulfamic and phosphotungstic acids. In addition, polyaniline films supplemented with poly (2-acrylamido-2-methyl-1-propanesulfonic) acid at various ratios were tested. Results showed that the NIH/3T3 cell line was able to adhere, proliferate and migrate on the pristine polyaniline films as well as those films doped with sulfamic and phosphotungstic acids; thus, utilization of said forms in biomedicine appears promising. Nevertheless, incorporating poly (2-acrylamido-2-methyl-1-propanesulfonic) acid altered the surface properties of the polyaniline films and significantly affected cell behavior. In order to reveal the crucial factor influencing the surface/cell interaction, cell behavior is discussed in the context of the surface energy of individual samples. It was clearly demonstrated that the lesser the difference between the surface energy of the sample and cell, the more cyto-compatible the surface is. PMID:27649159

  4. Bradykinin promotes TLR2 expression in human gingival fibroblasts.

    PubMed

    Gutiérrez-Venegas, Gloria; Arreguín-Cano, Juan Antonio

    2011-12-01

    Bradykinin (BK) is implicated in the sensation of pain, vasodilation, increases in vascular permeability and pathogenic processes associated with inflammation. Studies have shown that BK promotes the intracellular movement of calcium in human gingival fibroblasts by binding to the B2 receptor. In this study we investigated the effect of BK on regulation of Toll-like receptor 2 (TLR2) expression. Our results show that BK stimulates TLR2 receptor transcription and translation by activation of protein kinase C as well as AKT. Our study contributes important information on the regulation and expression of molecules that promote chronic inflammatory processes, which lead to periodontitis and consequently to loss of the dental organ.

  5. Regulation of fibronectin gene expression in cardiac fibroblasts by scleraxis.

    PubMed

    Bagchi, Rushita A; Lin, Justin; Wang, Ryan; Czubryt, Michael P

    2016-11-01

    The glycoprotein fibronectin is a key component of the extracellular matrix. By interacting with numerous matrix and cell surface proteins, fibronectin plays important roles in cell adhesion, migration and intracellular signaling. Up-regulation of fibronectin occurs in tissue fibrosis, and previous studies have identified the pro-fibrotic factor TGFβ as an inducer of fibronectin expression, although the mechanism responsible remains unknown. We have previously shown that a key downstream effector of TGFβ signaling in cardiac fibroblasts is the transcription factor scleraxis, which in turn regulates the expression of a wide variety of extracellular matrix genes. We noted that fibronectin expression tracked closely with scleraxis expression, but it was unclear whether scleraxis directly regulated the fibronectin gene. Here, we report that scleraxis acts via two E-box binding sites in the proximal human fibronectin promoter to govern fibronectin expression, with the second E-box being both sufficient and necessary for scleraxis-mediated fibronectin expression to occur. A combination of electrophoretic mobility shift and chromatin immunoprecipitation assays indicated that scleraxis interacted to a greater degree with the second E-box. Over-expression or knockdown of scleraxis resulted in increased or decreased fibronectin expression, respectively, and scleraxis null mice presented with dramatically decreased immunolabeling for fibronectin in cardiac tissue sections compared to wild-type controls. Furthermore, scleraxis was required for TGFβ-induced fibronectin expression: TGFβ lost its ability to induce fibronectin expression following scleraxis knockdown. Together, these results demonstrate a novel and required role for scleraxis in the regulation of cardiac fibroblast fibronectin gene expression basally or in response to TGFβ.

  6. 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

  7. EPAC expression and function in cardiac fibroblasts and myofibroblasts

    SciTech Connect

    Olmedo, Ivonne; Muñoz, Claudia; Guzmán, Nancy; Catalán, Mabel; Vivar, Raúl; Ayala, Pedro; Humeres, Claudio; Aránguiz, Pablo; García, Lorena; Velarde, Victoria; Díaz-Araya, Guillermo

    2013-10-15

    In the heart, cardiac fibroblasts (CF) and cardiac myofibroblasts (CMF) are the main cells responsible for wound healing after cardiac insult. Exchange protein activated by cAMP (EPAC) is a downstream effector of cAMP, and it has been not completely studied on CF. Moreover, in CMF, which are the main cells responsible for cardiac healing, EPAC expression and function are unknown. We evaluated in both CF and CMF the effect of transforming growth factor β1 (TGF-β1) on EPAC-1 expression. We also studied the EPAC involvement on collagen synthesis, adhesion, migration and collagen gel contraction. Method: Rat neonatal CF and CMF were treated with TGF-β1 at different times and concentrations. EPAC-1 protein levels and Rap1 activation were measured by western blot and pull down assay respectively. EPAC cellular functions were determined by adhesion, migration and collagen gel contraction assay; and collagen expression was determined by western blot. Results: TGF-β1 through Smad and JNK significantly reduced EPAC-1 expression in CF, while in CMF this cytokine increased EPAC-1 expression through ERK1/2, JNK, p38, AKT and Smad3. EPAC activation was able to induce higher Rap1-GTP levels in CMF than in CF. EPAC and PKA, both cAMP effectors, promoted CF and CMF adhesion on fibronectin, as well as CF migration; however, this effect was not observed in CMF. EPAC but not PKA activation mediated collagen gel contraction in CF, while in CMF both PKA and EPAC mediated collagen gel contraction. Finally, the EPAC and PKA activation reduced collagen synthesis in CF and CMF. Conclusion: TGF-β1 differentially regulates the expression of EPAC in CF and CMF; and EPAC regulates differentially CF and CMF functions associated with cardiac remodeling. - Highlights: • TGF-β1 regulates EPAC-1 expression in cardiac fibroblast and myofibroblast. • Rap-1GTP levels are higher in cardiac myofibroblast than fibroblast. • EPAC-1 controls adhesion, migration and collagen synthesis in cardiac

  8. Expression of the Saccharomyces cerevisiae glycoprotein invertase in mouse fibroblasts: glycosylation, secretion, and enzymatic activity

    SciTech Connect

    Bergh, M.L.E.; Cepko, C.L.; Wolf, D.; Robbins, P.W.

    1987-06-01

    Oligosaccharide processing is controlled by host- and protein-dependent factors. To increase our understanding of the relative contribution of those factors the authors studied the glycosylation of yeast invertase expressed in a heterologous system. Invertase synthesized in psi-2 cells (an NIH 3T3-derived packaging line) is secreted efficiently, enzymatically active, and heavily glycosylated. It was estimated that the protein contains 8 or 9 carbohydrate chains. Two classes can be observed, of an approximate size of 100-110 kDa and 115-130 kDa, respectively. The size differences are due to differences in glycosylation. The smaller class contains two high-mannose carbohydrate chains; the remainder is of the complex type, sialylated and most likely tri- or tetraantennary. This profile parallels the situation observed with invertase glycosylation in yeast, where 2 of 9 or 10 chains remain unprocessed. The larger size class of invertase expressed in mouse fibroblasts has a different profile, since it contains probably only complex-type glycans. There are no apparent differences, however, in the size of the protein backbone between the two size classes. When invertase is synthesized in the presence of the mannosidase inhibitor 1-deoxymannojirimycin, processing is blocked completely. The glucosidase inhibitor 1-deoxynojirimycin does not inhibit processing completely. The glycosylation inhibitor tunicamycin prevents secretion of invertase completely when cells are cultured at 37/sup 0/C. At 26/sup 0/C, however, nonglycosylated invertase can be detected in the medium. These data suggest that glycosylation of invertase seems to be essential for the early steps of the secretory pathway but is less critical for later events.

  9. Effects of fibroblast growth factor 2 on osteoblastic proliferation and differentiation by regulating bone morphogenetic protein receptor expression.

    PubMed

    Park, Jun-Beom

    2011-09-01

    Fibroblast growth factors (FGFs) are known to play a critical role in bone growth and development, affecting both osteogenesis and chondrogenesis. Fibroblast growth factor 2 (FGF-2) is produced intracellularly by osteoblasts and secreted into the surrounding matrix in bone.The dose-dependent effects of FGF-2 were tested to examine the relationship between FGF-2 and osteoblast proliferation and differentiation. Tests used included a cell viability test, an alkaline phosphatase activity test, and a Western blot analysis.Cultures growing in the presence of FGF-2 showed an increased value for 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay and a decreased value for alkaline phosphatase activity. Results of the Western blot analysis showed that the addition of FGF-2 seems to decrease osteocalcin and bone morphogenetic protein receptor IA.These data show that FGF-2 in the tested dosage within MC3T3-E1 cells seems to affect proliferation and differentiation. Results of the Western blot analysis may add some possible mechanisms, and it may be suggested that treatment of FGF-2 may have an influence on the expression of bone morphogenetic protein receptors in osteoprecursor cells. Further elucidation of the mechanisms related to this mechanism within the in vivo model may be necessary to ascertain greater detail.

  10. Averrhoa carambola L. peel extract suppresses adipocyte differentiation in 3T3-L1 cells.

    PubMed

    Rashid, Asyifah Mohamed; Lu, Kaihui; Yip, Yew Mun; Zhang, Dawei

    2016-02-01

    Obesity is associated with an increased risk of many chronic diseases. Recently, a growing body of evidence has shown that phytochemicals may inhibit adipogenesis and obesity. In this study, we report for the first time, the ability of Averrhoa carambola L. peel extract commonly known as star fruit (SFP) to effectively suppress adipocyte differentiation in 3T3-L1 preadipocytes and therefore, address it as a potential candidate to treat obesity and its related diseases. (-)-Epicatechin was identified as a bioactive compound likely responsible for this suppression. As the genetic expression studies revealed that the adipogenic activity of SFP extract was due to the simultaneous downregulation of the C/EBPα and PPARγ as well as the upregulation of PPARα receptor genes, a detailed computational docking study was also elucidated to reveal the likely binding mode of (-)-epicatechin to the receptor of interest, accounting for the likely mechanism that results in the overall suppression of adipocyte differentiation.

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

    PubMed

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

    2013-05-01

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

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

    PubMed Central

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

    2013-01-01

    Synthetic glucocorticoids are potent anti-inflammatory drugs but show dose-dependent metabolic side effects such as the development of insulin resistance and obesity. The precise mechanisms involved in these glucocorticoid-induced side effects, and especially the participation of adipose tissue in this are not completely understood. We used a combination of transcriptomics, antibody arrays and bioinformatics approaches to characterize prednisolone-induced alterations in gene expression and adipokine secretion, which could underlie metabolic dysfunction in 3T3-L1 adipocytes. Several pathways, including cytokine signalling, Akt signalling, and Wnt signalling were found to be regulated at multiple levels, showing that these processes are targeted by prednisolone. These results suggest that mechanisms by which prednisolone induce insulin resistance include dysregulation of wnt signalling and immune response processes. These pathways may provide interesting targets for the development of improved glucocorticoids. PMID:23506355

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

    PubMed

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

    2010-06-24

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

  14. Melatonin Suppresses Autophagy Induced by Clinostat in Preosteoblast MC3T3-E1 Cells.

    PubMed

    Yoo, Yeong-Min; Han, Tae-Young; Kim, Han Sung

    2016-04-08

    Microgravity exposure can cause cardiovascular and immune disorders, muscle atrophy, osteoporosis, and loss of blood and plasma volume. A clinostat device is an effective ground-based tool for simulating microgravity. This study investigated how melatonin suppresses autophagy caused by simulated microgravity in preosteoblast MC3T3-E1 cells. In preosteoblast MC3T3-E1 cells, clinostat rotation induced a significant time-dependent increase in the levels of the autophagosomal marker microtubule-associated protein light chain (LC3), suggesting that autophagy is induced by clinostat rotation in these cells. Melatonin treatment (100, 200 nM) significantly attenuated the clinostat-induced increases in LC3 II protein, and immunofluorescence staining revealed decreased levels of both LC3 and lysosomal-associated membrane protein 2 (Lamp2), indicating a decrease in autophagosomes. The levels of phosphorylation of mammalian target of rapamycin (p-mTOR) (Ser2448), phosphorylation of extracellular signal-regulated kinase (p-ERK), and phosphorylation of serine-threonine protein kinase (p-Akt) (Ser473) were significantly reduced by clinostat rotation. However, their expression levels were significantly recovered by melatonin treatment. Also, expression of the Bcl-2, truncated Bid, Cu/Zn- superoxide dismutase (SOD), and Mn-SOD proteins were significantly increased by melatonin treatment, whereas levels of Bax and catalase were decreased. The endoplasmic reticulum (ER) stress marker GRP78/BiP, IRE1α, and p-PERK proteins were significantly reduced by melatonin treatment. Treatment with the competitive melatonin receptor antagonist luzindole blocked melatonin-induced decreases in LC3 II levels. These results demonstrate that melatonin suppresses clinostat-induced autophagy through increasing the phosphorylation of the ERK/Akt/mTOR proteins. Consequently, melatonin appears to be a potential therapeutic agent for regulating microgravity-related bone loss or osteoporosis.

  15. Melatonin Suppresses Autophagy Induced by Clinostat in Preosteoblast MC3T3-E1 Cells

    PubMed Central

    Yoo, Yeong-Min; Han, Tae-Young; Kim, Han Sung

    2016-01-01

    Microgravity exposure can cause cardiovascular and immune disorders, muscle atrophy, osteoporosis, and loss of blood and plasma volume. A clinostat device is an effective ground-based tool for simulating microgravity. This study investigated how melatonin suppresses autophagy caused by simulated microgravity in preosteoblast MC3T3-E1 cells. In preosteoblast MC3T3-E1 cells, clinostat rotation induced a significant time-dependent increase in the levels of the autophagosomal marker microtubule-associated protein light chain (LC3), suggesting that autophagy is induced by clinostat rotation in these cells. Melatonin treatment (100, 200 nM) significantly attenuated the clinostat-induced increases in LC3 II protein, and immunofluorescence staining revealed decreased levels of both LC3 and lysosomal-associated membrane protein 2 (Lamp2), indicating a decrease in autophagosomes. The levels of phosphorylation of mammalian target of rapamycin (p-mTOR) (Ser2448), phosphorylation of extracellular signal-regulated kinase (p-ERK), and phosphorylation of serine-threonine protein kinase (p-Akt) (Ser473) were significantly reduced by clinostat rotation. However, their expression levels were significantly recovered by melatonin treatment. Also, expression of the Bcl-2, truncated Bid, Cu/Zn- superoxide dismutase (SOD), and Mn-SOD proteins were significantly increased by melatonin treatment, whereas levels of Bax and catalase were decreased. The endoplasmic reticulum (ER) stress marker GRP78/BiP, IRE1α, and p-PERK proteins were significantly reduced by melatonin treatment. Treatment with the competitive melatonin receptor antagonist luzindole blocked melatonin-induced decreases in LC3 II levels. These results demonstrate that melatonin suppresses clinostat-induced autophagy through increasing the phosphorylation of the ERK/Akt/mTOR proteins. Consequently, melatonin appears to be a potential therapeutic agent for regulating microgravity-related bone loss or osteoporosis. PMID:27070587

  16. Honokiol enhances adipocyte differentiation by potentiating insulin signaling in 3T3-L1 preadipocytes.

    PubMed

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

    2011-07-01

    Adipose tissue plays an essential role in energy homeostasis as a metabolic and endocrine organ. Accordingly, adipocytes are emerging as a major drug target for obesity and obesity-mediated metabolic syndrome. Dysfunction of enlarged adipocytes in obesity is involved in obesity-mediated metabolic syndrome. Adipocytokines, such as adiponectin released from small adipocytes, are able to prevent these disorders. In this study, we found that honokiol, an ingredient of Magnolia officinalis used in traditional Chinese and Japanese medicines, enhanced adipocyte differentiation in 3T3-L1 preadipocytes. Oil Red O staining showed that treatment with honokiol in the presence of insulin dose-dependently increased lipid accumulation in 3T3-L1 preadipoyctes although its activity was weak compared with rosiglitazone. During adipocyte differentiation, the expression of peroxisome proliferator-activated receptor γ2 (PPARγ2) mRNA and PPARγ target genes such as adipocyte protein 2 (aP2), adiponectin, and GLUT4 was induced by treatment with 10 μM honokiol. However, honokiol failed to show direct binding to the PPARγ ligand-binding domain in vitro. In preadipocytes, treatment with honokiol in the presence of insulin increased the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 protein and Akt protein, early insulin signaling pathways related to adipocyte differentiation, compared with insulin-only treatment. Taken together, our results suggest that honokiol promotes adipocyte differentiation through increased expression of PPARγ2 mRNA and potentiation of insulin signaling pathways such as the Ras/ERK1/2 and phosphoinositide-3-kinase (PI3K)/Akt signaling pathways.

  17. Monascus ruber-Fermented Buckwheat (Red Yeast Buckwheat) Suppresses Adipogenesis in 3T3-L1 Cells.

    PubMed

    Hong, Heeok; Park, Jiyoung; Lumbera, Wenchie L; Hwang, Seong Gu

    2017-03-23

    Although various treatments have been used for weight loss to date, obese people rarely have safe and effective treatment options. Therefore, the antiobesity effects of several natural compounds are being actively investigated. This study was conducted to investigate the antiadipogenic effects of Monascus ruber-fermented Fagopyrum esculentum (red yeast buckwheat, RYB) in 3T3-L1 cells. We assessed the intracellular lipid content and adipocyte differentiation by oil red O staining and the expression of genes and proteins associated with adipocyte differentiation by reverse transcription-polymerase chain reaction and western blotting in 3T3-L1 cells. RYB dose dependently inhibited 3T3-L1 cell differentiation at concentrations of 50-800 μg/mL, without cytotoxic effects. It also suppressed the expression of adipogenic transcription factors, including peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α, and adipocyte-specific genes, such as adipocyte fatty acid-binding protein (aP2), fatty acid synthase, and leptin, during preadipocyte differentiation into adipocytes. Furthermore, RYB reduced cyclin-dependent kinase 2 and cyclin expression and increased p21 and p27 expression, thus causing cell cycle arrest at the G1/S phase. Collectively, these results suggest that RYB may be an effective nutraceutical for weight loss as indicated by its ability to suppress adipogenesis-specific gene expression and cause cell cycle arrest at the G1/S interphase.

  18. Cellular retinol-binding protein-1 is transiently expressed in granulation tissue fibroblasts and differentially expressed in fibroblasts cultured from different organs.

    PubMed Central

    Xu, G.; Redard, M.; Gabbiani, G.; Neuville, P.

    1997-01-01

    We have reported that cellular retinol-binding protein-1 (CRBP-1) is transiently expressed by arterial smooth muscle cells during experimental intimal repair (P. Neuville, A. Geinoz, G. Benzonana, M. Redard, F. Gabbiani, P. Ropraz, G. Gabbiani: Am J Pathol 1997, 150:509-521). We have examined here the expression of CRBP-1 during wound healing after a full-thickness rat skin wound. CRBP-1 was transiently expressed by a significant proportion of fibroblastic cells including myofibroblasts. Expression started 4 days after wounding, reached a maximum at 12 days, and persisted up to 30 days when a scar was formed. After wound closure, most CRBP-1-containing fibroblastic cells underwent apoptosis. We have further investigated CRBP-1 expression in rat fibroblasts cultured from different organs. CRBP-1 was abundant in lung and heart fibroblasts and was detected in decreasing amounts in muscle, tendon, subcutaneous tissue, and granulation tissue fibroblasts. Dermis fibroblasts contained no detectable levels of CRBP-1. All-trans retinoic acid and transforming growth factor-beta1 inhibited cell proliferation and increased CRBP-1 expression in fibroblastic populations except dermis fibroblasts. We demonstrate that during granulation tissue formation a subpopulation of fibroblastic cells express CRBP-1 de novo. We also demonstrate that CRBP-1 expression by fibroblasts is regulated in vitro by retinoic acid and transforming growth factor-beta1. Our results suggest that CRBP-1 and possibly retinoic acid play a role in the evolution of granulation tissue. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 7 PMID:9403724

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

    PubMed Central

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

    2014-01-01

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

  20. 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.

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

    SciTech Connect

    Eda, Homare; Aoki, Katsuhiko; Marumo, Keishi; Fujii, Katsuyuki; Ohkawa, Kiyoshi

    2008-02-08

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

  2. 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-α, PPAR-γ, 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-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), 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-α, PPAR-γ, FAS, STAT-3 and STAT-5.

  3. Smad gene expression in pulmonary fibroblasts: indications for defective ECM repair in COPD

    PubMed Central

    Zandvoort, Andre; Postma, Dirkje S; Jonker, Marnix R; Noordhoek, Jacobien A; Vos, Johannes TWM; Timens, Wim

    2008-01-01

    Background Chronic Obstructive Pulmonary Disease (COPD) is characterized by defective extracellular matrix (ECM) turnover as a result of prolonged cigarette smoking. Fibroblasts have a central role in ECM turnover. The TGFβ induced Smad pathway provides intracellular signals to regulate ECM production. We address the following hypothesis: fibroblasts have abnormal expression of genes in the Smad pathway in COPD, resulting in abnormal proteoglycan modulation, the ground substance of ECM. Methods We compared gene expression of the Smad pathway at different time points after stimulation with TGFβ, TNF or cigarette smoke extract (CSE) in pulmonary fibroblasts of GOLD stage II and IV COPD patients, and controls. Results Without stimulation, all genes were similarly expressed in control and COPD fibroblasts. TGFβ stimulation: downregulation of Smad3 and upregulation of Smad7 occurred in COPD and control fibroblasts, indicating a negative feedback loop upon TGFβ stimulation. CSE hardly influenced gene expression of the TGFβ-Smad pathway in control fibroblasts, whereas it reduced Smad3 and enhanced Smad7 gene expression in COPD fibroblasts. Furthermore, decorin gene expression decreased by all stimulations in COPD but not in control fibroblasts. Conclusion Fibroblasts of COPD patients and controls differ in their regulation of the Smad pathway, the contrast being most pronounced under CSE exposure. This aberrant responsiveness of COPD fibroblasts to CSE might result in an impaired tissue repair capability and is likely important with regard to the question why only a subset of smokers demonstrates an excess ECM destruction under influence of cigarette smoking. PMID:19087346

  4. 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 Col1α1. - 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 Col1α1. 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 Collα1 gene expression.

  5. Expression of biologically recombinant human acidic fibroblast growth factor in Arabidopsis thaliana seeds via oleosin fusion technology.

    PubMed

    Yang, Jing; Guan, Lili; Guo, Yongxin; Du, Linna; Wang, Fawei; Wang, Yanfang; Zhen, Lu; Wang, Qingman; Zou, Deyi; Chen, Wei; Yu, Lei; Li, Haiyan; Li, Xiaokun

    2015-07-15

    The potential of oleosins to act as carriers for recombinant foreign proteins in plant cells has been established. Using the oleosin fusion technology, the protein can be targeted to oil bodies in oilseeds by fusing it to the N- or C-terminus of oleosin. In this study, aFGF was expressed in Arabidopsis thaliana seeds via oleosin fusion technology. A plant-preferred aFGF gene was synthesized by optimizing codon usage and was fused to the C-terminus of the A. thaliana 18.5kDa oleosin gene. The fusion gene was driven by the phaseolin promoter to confer seed-specific expression of the human acidic fibroblast growth factor in A. thaliana. The T-DNA region of the recombinant plasmid pKO-aFGF was introduced into the genome of Arabidopsis thaliana by the floral dip method. The aFGF protein expression was confirmed by SDS-PAGE and western blotting. The biological activity showed that oil bodies fused to aFGF stimulated NIH/3T3 cell proliferation activity.

  6. 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

    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.

  7. 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.

  8. Fibroblast Growth Factor Receptor-4 and Prostate Cancer Progression

    DTIC Science & Technology

    2006-10-01

    variant arising from a cryptic promoter (ptd- FGFR-4) is expressed in pituitary adenomas and that expression of this variant resulted in increased inva...siveness of pituitary cells. Expression of the ptd-FGFR- 4 variant decreased adhesion to collagen IV in pituitary cells and NIH3T3 fibroblasts...formation of NCAM and N- cadherin complexes in pituitary cells which could affect adhesion to collagen IV. On the other hand, Coppolino and Dedhar [37

  9. Fibroblast Growth Factor Receptor-4 and Prostate Cancer Progression

    DTIC Science & Technology

    2007-10-01

    al. [24] have reported that a cytoplasmic FGFR-4 variant arising from a cryptic promoter (ptd- FGFR-4) is expressed in pituitary adenomas and that...expression of this variant resulted in increased inva- siveness of pituitary cells. Expression of the ptd-FGFR- 4 variant decreased adhesion to collagen...IV in pituitary cells and NIH3T3 fibroblasts compared to full length FGFR-4 in a ligand independentmanner. These authors provide evidence that

  10. 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

  11. Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP.

    PubMed Central

    Feig, L A; Cooper, G M

    1988-01-01

    Substitution of asparagine for serine at position 17 decreased the affinity of rasH p21 for GTP 20- to 40-fold without significantly affecting its affinity for GDP. Transfection of NIH 3T3 cells with a mammalian expression vector containing the Asn-17 rasH gene and a Neor gene under the control of the same promoter yielded only a small fraction of the expected number of G418-resistant colonies, indicating that expression of Asn-17 p21 inhibited cell proliferation. The inhibitory effect of Asn-17 p21 required its localization to the plasma membrane and was reversed by coexpression of an activated ras gene, indicating that the mutant p21 blocked the endogenous ras function required for NIH 3T3 cell proliferation. NIH 3T3 cells transformed by v-mos and v-raf, but not v-src, were resistant to inhibition by Asn-17 p21, indicating that the requirement for normal ras function can be bypassed by these cytoplasmic oncogenes. The Asn-17 mutant represents a novel reagent for the study of ras function by virtue of its ability to inhibit cellular ras activity in vivo. Since this phenotype is likely associated with the preferential affinity of the mutant protein for GDP, analogous mutations might also yield inhibitors of other proteins whose activities are regulated by guanine nucleotide binding. Images PMID:3145408

  12. Cocoa tea (Camellia ptilophylla) water extract inhibits adipocyte differentiation in mouse 3T3-L1 preadipocytes

    PubMed Central

    Li, Kai Kai; Liu, Chuek Lun; Shiu, Hoi Ting; Wong, Hing Lok; Siu, Wing Sum; Zhang, Cheng; Han, Xiao Qiang; Ye, Chuang Xing; Leung, Ping Chung; Ko, Chun Hay

    2016-01-01

    Cocoa tea (Camellia ptilophylla) is a naturally decaffeinated tea plant. Previously we found that cocoa tea demonstrated a beneficial effect against high-fat diet induced obesity, hepatic steatosis, and hyperlipidemia in mice. The present study aimed to investigate the anti-adipogenic effect of cocoa tea in vitro using preadipocytes 3T3-L1. Adipogenic differentiation was confirmed by Oil Red O stain, qPCR and Western blot. Our results demonstrated that cocoa tea significantly inhibited triglyceride accumulation in mature adipocytes in a dose-dependent manner. Cocoa tea was shown to suppress the expressions of key adipogenic transcription factors, including peroxisome proliferator-activated receptor gamma (PPAR γ) and CCAAT/enhancer binding protein (C/EBP α). The tea extract was subsequently found to reduce the expressions of adipocyte-specific genes such as sterol regulatory element binding transcription factor 1c (SREBP-1c), fatty acid synthase (FAS), Acetyl-CoA carboxylase (ACC), fatty acid translocase (FAT) and stearoylcoenzyme A desaturase-1 (SCD-1). In addition, JNK, ERK and p38 phosphorylation were inhibited during cocoa tea inhibition of 3T3-L1 adipogenic differentiation. Taken together, this is the first study that demonstrates cocoa tea has the capacity to suppress adipogenesis in pre-adipocyte 3T3-L1 similar to traditional green tea PMID:26833256

  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-γ 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. Suppressive Effects of Barley β-Glucans with Different Molecular Weight on 3T3-L1 Adipocyte Differentiation.

    PubMed

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

    2016-03-01

    In this study, 2 β-glucans with different molecular weight were prepared and purified from hull-less barley bran. The aim was to evaluate their effects on the differentiation of 3T3-L1 pre-adipocytes. Results showed that barley β-glucans inhibited the differentiation of 3T3-L1 pre-adipocytes induced by differentiation medium in a dose-dependent manner, the suppressive effect of high-molecular-weight barley β-glucans (552 kDa, BGH) was stronger (P < 0.05) than that of low-molecular-weight barley β-glucan (32 kDa, BGL), evidenced by the significantly decrease (P < 0.05) of Oil-red O staining and intracellular triglyceride content in the mature adipocytes. Besides, gene expression analysis and Western Blot analysis revealed that both BGH and BGL inhibited the mRNA and protein levels of adipogenesis related transcription factors peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT-enhancer-binding protein α (C/EBPα) which are principal regulators of adipogenesis. Furthermore, the mRNA and protein expression levels of PPARγ target genes in adipose tissue including adipocyte fatty acid binding protein (ap2), lipoprotein lipase (LPL), uncoupling protein-2 (UCP-2), and glucose-transporter 4 (Glut4) in 3T3-L1 cells was also markedly downregulated (P < 0.05). These findings were anticipated to help develop barley β-glucans based functional food for the management of obesity.

  15. Cocoa tea (Camellia ptilophylla) water extract inhibits adipocyte differentiation in mouse 3T3-L1 preadipocytes.

    PubMed

    Li, Kai Kai; Liu, Chuek Lun; Shiu, Hoi Ting; Wong, Hing Lok; Siu, Wing Sum; Zhang, Cheng; Han, Xiao Qiang; Ye, Chuang Xing; Leung, Ping Chung; Ko, Chun Hay

    2016-02-01

    Cocoa tea (Camellia ptilophylla) is a naturally decaffeinated tea plant. Previously we found that cocoa tea demonstrated a beneficial effect against high-fat diet induced obesity, hepatic steatosis, and hyperlipidemia in mice. The present study aimed to investigate the anti-adipogenic effect of cocoa tea in vitro using preadipocytes 3T3-L1. Adipogenic differentiation was confirmed by Oil Red O stain, qPCR and Western blot. Our results demonstrated that cocoa tea significantly inhibited triglyceride accumulation in mature adipocytes in a dose-dependent manner. Cocoa tea was shown to suppress the expressions of key adipogenic transcription factors, including peroxisome proliferator-activated receptor gamma (PPAR γ) and CCAAT/enhancer binding protein (C/EBP α). The tea extract was subsequently found to reduce the expressions of adipocyte-specific genes such as sterol regulatory element binding transcription factor 1c (SREBP-1c), fatty acid synthase (FAS), Acetyl-CoA carboxylase (ACC), fatty acid translocase (FAT) and stearoylcoenzyme A desaturase-1 (SCD-1). In addition, JNK, ERK and p38 phosphorylation were inhibited during cocoa tea inhibition of 3T3-L1 adipogenic differentiation. Taken together, this is the first study that demonstrates cocoa tea has the capacity to suppress adipogenesis in pre-adipocyte 3T3-L1 similar to traditional green tea.

  16. Transformation by viral and cellular oncogenes of a mouse BALB/3T3 cell mutant resistant to transformation by chemical carcinogens

    SciTech Connect

    Ono, M.; Yakushinji, M.; Segawa, K.; Kuwano, M.

    1988-10-01

    The mouse cell line MO-5 is resistant to transformation by various chemical carcinogens and also by UV irradiation. Northern (RNA) blot analysis showed active expression of ras and myc genes in MO-5 and BALB/3T3 cells. The effect of transfection of various oncogenes on transformation was compared in MO-5 cells and parental BALB/3T3 cells. Activated c-H-ras, c-N-ras, and v-mos gene induced transformation foci of MO-5 and BALB/3T3. Introduction of the polyomavirus middle T-antigen (mTag) or the Rous sarcoma virus-related oncogene v-src, however, efficiently transformed BALB/3T3 but not MO-5 cells. Expression and phosphorylation of mTag and the associated c-src proteins were observed in mTag-transfected clones of MO-5 as in BALB/3T3 and phosphorylation of the src protein was observed in v-src-transfected BALB/3T3 and MO-5 clones. Hybrids between mTag- or v-src-induced transformants of BALB/3T3 and untransformed MO-5 maintained the transformation phenotype, suggesting that no dominant suppressor of transformation exists in MO-5. A hybrid clone between BALB/3T3 and MO-5 induced efficient transformation foci after transfection with the mTag gene, suggesting that the deficient transformation phenotype of MO-5 was recessive. Instead, some other alteration of MO-5, plausibly membrane function, might lead to abortive transformation by chemical carcinogens and also by mTag and the v-src gene product.

  17. Assessment of the phototoxic hazard of some essential oils using modified 3T3 neutral red uptake assay.

    PubMed

    Dijoux, Nathalie; Guingand, Yannick; Bourgeois, Caroline; Durand, Sandrine; Fromageot, Claude; Combe, Corinne; Ferret, Pierre-Jacques

    2006-06-01

    When substances are developed in the aim to be a constituent of personal care products, and to be applied on the skin, it is necessary to carry out an assessment of potential phototoxic hazard. Phototoxicity is skin reaction caused by concurrent topical or systemic exposure to specific molecule and ultraviolet radiation. Most phototoxic compounds absorb energy particularly from UVA light leading to the generation of activated derivatives which can induce cellular damage. This type of adverse cutaneous response can be reproduced in vitro using different models of phototoxicity such as the validated 3T3 Neutral Red Uptake (NRU) phototoxicity assay. In the present study we utilised two different cell lines (the murine fibroblastic cell line 3T3 and the rabbit cornea derived cell line SIRC) to compare the photo-irritation potential of a strong phototoxic compound, chlorpromazine, to a weaker composite, such as 8-methoxypsoralen and Bergamot oil. After comparison of the different systems, five other essential oils were tested with both cell lines. Cellular damage was evaluated by the NRU cytotoxicity test or by MTT conversion test.

  18. EGF raises cytosolic Ca sup 2+ in A431 and Swiss 3T3 cells by a dual mechanism

    SciTech Connect

    Pandiella, A.; Malgaroli, A.; Meldolesi, J.; Vicentini, L.M. )

    1987-05-01

    The changes in Ca{sup 2+} homeostasis and phosphoinositide hydrolysis induced by EGF were studied in human epidermoid carcinoma A431 cells both when attached to a substratum and after detachment and suspension. The cytosolic Ca{sup 2+} concentration was measured by the conventional fluorimetric technique, using the specific probe, quin2, as well as by a new microscopic technique in which single cells are investigated after loading with another probe, fura-2. EGF applied in the complete, Ca{sup 2+}-containing medium caused a rapid rise in the cytosolic {sup 45}Ca{sup 2+} concentration, that remained elevated for several minutes. In Ca{sup 2+}-free, EGTA-containing medium, part of this response persisted, as revealed by quin2 results in suspended cells and microscopic results with fura-2. These results, as well as additional microscopic fura-2 results in Swiss 3T3 fibroblasts, demonstrate that the Ca{sup 2+} signal elicited by EGF is due to two components: redistribution from an intracellular store and stimulated influx across the plasmalemma. This latter process was not detected in 3T3 cells treated with either PDGF or bombesin. It is therefore suggested that the {sup 45}Ca{sup 2+} influx effect of EGF is under the control of a separate, as yet unidentified mechanism.

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

    PubMed Central

    Si, Yaguang; Shi, Hai; Lee, Kyongbum

    2009-01-01

    Background Increasing energy expenditure at the cellular level offers an attractive option to limit adiposity and improve whole body energy balance. In vivo and in vitro observations have correlated mitochondrial uncoupling protein-1 (UCP1) expression with reduced white adipose tissue triglyceride (TG) content. The metabolic basis for this correlation remains unclear. Methodology/Principal Findings This study tested the hypothesis that mitochondrial uncoupling requires the cell to compensate for the decreased oxidation phosphorylation efficiency by up-regulating lactate production, thus redirecting carbon flux away from TG synthesis. Metabolic flux analysis was used to characterize the effects of non-lethal, long-term mitochondrial uncoupling (up to 18 days) on the pathways of intermediary metabolism in differentiating 3T3-L1 adipocytes. Uncoupling was induced by forced expression of UCP1 and chemical (FCCP) treatment. Chemical uncoupling significantly decreased TG content by ca. 35%. A reduction in the ATP level suggested diminished oxidative phosphorylation efficiency in the uncoupled adipocytes. Flux analysis estimated significant up-regulation of glycolysis and down-regulation of fatty acid synthesis, with chemical uncoupling exerting quantitatively larger effects. Conclusions/Significance The results of this study support our hypothesis regarding uncoupling-induced redirection of carbon flux into glycolysis and lactate production, and suggest mitochondrial proton translocation as a potential target for controlling adipocyte lipid metabolism. PMID:19746157

  20. Irradiated fibroblasts promote epithelial–mesenchymal transition and HDGF expression of esophageal squamous cell carcinoma

    SciTech Connect

    Bao, Ci-Hang; Wang, Xin-Tong; Ma, Wei; Wang, Na-Na; Nesa, Effat un; Wang, Jian-Bo; Wang, Cong; Jia, Yi-Bin; Wang, Kai; Tian, Hui; Cheng, Yu-Feng

    2015-03-06

    Recent evidence suggested that nonirradiated cancer-associated fibroblasts (CAFs) promoted aggressive phenotypes of cancer cells through epithelial–mesenchymal transition (EMT). Hepatoma-derived growth factor (HDGF) is a radiosensitive gene of esophageal squamous cell carcinoma (ESCC). This study aimed to investigate the effect of irradiated fibroblasts on EMT and HDGF expression of ESCC. Our study demonstrated that coculture with nonirradiated fibroblasts significantly increased the invasive ability of ESCC cells and the increased invasiveness was further accelerated when they were cocultured with irradiated fibroblasts. Scattering of ESCC cells was also accelerated by the supernatant from irradiated fibroblasts. Exposure of ESCC cells to supernatant from irradiated fibroblasts resulted in decreased E-cadherin, increased vimentin in vitro and β-catenin was demonstrated to localize to the nucleus in tumor cells with irradiated fibroblasts in vivo models. The expression of HDGF and β-catenin were increased in both fibroblasts and ESCC cells of irradiated group in vitro and in vivo models. Interestingly, the tumor cells adjoining the stromal fibroblasts displayed strong nuclear HDGF immunoreactivity, which suggested the occurrence of a paracrine effect of fibroblasts on HDGF expression. These data suggested that irradiated fibroblasts promoted invasion, growth, EMT and HDGF expression of ESCC. - Highlights: • Irradiated CAFs accelerated invasiveness and scattering of ESCC cell lines. • Irradiated CAFs promoted EMT of ESCC cells. • Irradiated fibroblasts induced nuclear β-catenin relocalization in ESCC cells. • Irradiated fibroblasts increased HDGF expression in vitro and in vivo.

  1. ClC-3 Promotes Osteogenic Differentiation in MC3T3-E1 Cell After Dynamic Compression.

    PubMed

    Wang, Dawei; Wang, Hao; Gao, Feng; Wang, Kun; Dong, Fusheng

    2017-06-01

    ClC-3 chloride channel has been proved to have a relationship with the expression of osteogenic markers during osteogenesis, persistent static compression can upregulate the expression of ClC-3 and regulate osteodifferentiation in osteoblasts. However, there was no study about the relationship between the expression of ClC-3 and osteodifferentiation after dynamic compression. In this study, we applied dynamic compression on MC3T3-E1 cells to detect the expression of ClC-3, runt-related transcription factor 2 (Runx2), bone morphogenic protein-2 (BMP-2), osteopontin (OPN), nuclear-associated antigen Ki67 (Ki67), and proliferating cell nuclear antigen (PCNA) in biopress system, then we investigated the expression of these genes after dynamic compression with Chlorotoxin (specific ClC-3 chloride channel inhibitor) added. Under transmission electron microscopy, there were more cell surface protrusions, rough surfaced endoplasmic reticulum, mitochondria, Golgi apparatus, abundant glycogen, and lysosomes scattered in the cytoplasm in MC3T3-E1 cells after dynamic compression. The nucleolus was more obvious. We found that ClC-3 was significantly up-regulated after dynamic compression. The compressive force also up-regulated Runx2, BMP-2, and OPN after dynamic compression for 2, 4 and 8 h. The proliferation gene Ki67 and PCNA did not show significantly change after dynamic compression for 8 h. Chlorotoxin did not change the expression of ClC-3 but reduced the expression of Runx2, BMP-2, and OPN after dynamic compression compared with the group without Cltx added. The data from the current study suggested that ClC-3 may promotes osteogenic differentiation in MC3T3-E1 cell after dynamic compression. J. Cell. Biochem. 118: 1606-1613, 2017. © 2016 Wiley Periodicals, Inc.

  2. Phosphorylation of the growth arrest-specific protein Gas2 is coupled to actin rearrangements during Go-->G1 transition in NIH 3T3 cells

    PubMed Central

    1994-01-01

    Growth arrest-specific (Gas2) protein has been shown to be a component of the microfilament system, that is highly expressed in growth arrested mouse and human fibroblasts and is hyperphosphorylated upon serum stimulation of quiescent cells. (Brancolini, C., S. Bottega, and C. Schneider. 1992. J. Cell Biol. 117:1251-1261). In this study we demonstrate that the kinetics of Gas2 phosphorylation, during Go-->G1 transition, as induced by addition of 20% FCS to serum starved NIH 3T3 cells, is temporally coupled to the reorganization of actin cytoskeleton. To better dissect the relationship between Gas2 phosphorylation and the modification of the microfilament architecture we used specific stimuli for both membrane ruffling (PDGF and PMA) and stress fiber formation (L-alpha-lysophosphatidic acid LPA) (Ridley, A. J., and A. Hall. 1992. Cell. 70:389-399). All of them, similarly to 20% FCS, are able to downregulate Gas2 biosynthesis. PDGF and PMA induce Gas2 hyperphosphorylation that is temporally coupled with the appearance of membrane ruffling where Gas2 localizes. On the other hand LPA, a specific stimulus for stress fiber formation, fails to induce a detectable Gas2 hyperphosphorylation. Thus, Gas2 hyperphosphorylation is specifically correlated with the formation of membrane ruffling possibly implying a role of Gas2 in this process. PMID:8120096

  3. Exendin-4 promotes proliferation and differentiation of MC3T3-E1 osteoblasts by MAPKs activation.

    PubMed

    Feng, Yingyu; Su, Lei; Zhong, Xing; Guohong, Wei; Xiao, Haipeng; Li, Yanbing; Xiu, Lingling

    2016-04-01

    Glucagon-like peptide-1 (GLP1) and its receptor agonist have been previously reported to play a positive role in bone metabolism in aged ovariectomized rats and insulin-resistant models. However, whether GLP1 has a direct effect on the proliferation and differentiation of osteoblasts or any cellular mechanism for this potential role is unknown. We examined the effects of the GLP1 receptor agonist exendin-4 on the proliferation, differentiation, and mineralization of mouse osteoblastic MC3T3-E1 cells. GLP1 receptor was detected in MC3T3-E1 cells by polymerase chain reaction (PCR) and Western blot assay. Cell proliferation was assessed using MTT assay, revealing that exendin-4 increased cell proliferation at effective concentrations between 10(-10) and 10(-5) M. Quantitative PCR analysis showed that exendin-4 increased the mRNA expression of the differentiation markers alkaline phosphatase (ALP), collagen-1 (COL1), osteocalcin (OC), and runt-related transcription factor 2 (RUNX2) under osteogenic conditions. Alizarin red staining confirmed that 10(-7) M exendin-4 increased osteoblast mineralization by 18.7%. Exendin-4 upregulated the phosphorylation of ERK1/2, p38, and JNK, with the peak effect at 1.5 h in the Western blot analysis. The use of selective MAPK inhibitors, namely PD98059, SB203580, and SP600125, blocked the effects of exendin-4 on kinase activation (ERK1/2, p38, and JNK), as well as cell proliferation and differentiation in MC3T3-E1 cells. These findings demonstrate that exendin-4 promotes both the proliferation and differentiation of preosteoblasts MC3T3-E1 via activation of the MAPK pathway.

  4. Sphingosine-1-phosphate inhibits the adipogenic differentiation of 3T3-L1 preadipocytes.

    PubMed

    Moon, Myung-Hee; Jeong, Jae-Kyo; Lee, You-Jin; Seol, Jae-Won; Park, Sang-Youel

    2014-10-01

    Sphingosine-1-phosphate (S1P) is a pluripotent lipid mediator that transmits signals through G-protein-coupled receptors to control diverse biological processes. The novel biological activity of S1P in the adipogenesis of 3T3-L1 preadipocytes was identified in the present study. S1P significantly decreased lipid accumulation in maturing preadipocytes in a dose‑dependent manner. In order to understand the anti‑adipogenic effects of S1P, preadipocytes were treated with S1P, and the change in the expression of several adipogenic transcription factors and enzymes was investigated using quantitative RT-PCR. S1P downregulated the transcriptional levels of the peroxisome proliferator-activated receptor γ, CCAAT/enhancer binding proteins and adiponectin, which are markers of adipogenic differentiation. The effects of S1P on the levels of mitogen‑activated protein kinase (MAPK) signals in preadipocytes were also investigated. The activation of JNK and p38 were downregulated by S1P treatment in human preadipocytes. In conclusion, the results of this study suggest that S1P alters fat mass by directly affecting adipogenesis. This is mediated by the downregulation of adipogenic transcription factors and by inactivation of the JNK and p38 MAPK pathways. Thus, selective targeting of the S1P receptors and sphingosine kinases may have clinical applications for the treatment of obesity.

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

    PubMed

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

    2013-07-01

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

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

    SciTech Connect

    He, Yonghan; Li, Ying; Zhang, Shuocheng; Perry, Ben; Zhao, Tiantian; Wang, Yanwen; 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 γ (PPAR{sub γ}) and CCAAT element binding protein α (C/EBP{sub α}), 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 β-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.

  7. Advanced oxidation protein products inhibit differentiation and activate inflammation in 3T3-L1 preadipocytes.

    PubMed

    Zhou, Qiu Gen; Peng, Xin; Hu, Li Li; Xie, Di; Zhou, Min; Hou, Fan Fan

    2010-10-01

    Accumulation of advanced oxidation protein products (AOPPs) is prevalent in metabolic syndromes, a condition with impaired preadipocytes differentiation. In the present study, we tested the hypothesis that AOPPs disturb preadipocyte differentiation. Exposure of 3T3-L1 preadipocytes to increased levels of AOPPs inhibited accumulation of intracellular triglyceride and decreased the expression of the essential markers of matured adipocytes, such as adipocyte fatty-acid-binding protein (aP2), CAAT/enhancer-binding protein (C/EBP)-alpha, and peroxisome proliferator-activated receptor (PPAR)-gamma, in response to standard adipogenic induction. Inhibitory effects of AOPPs on preadipocytes differentiation was time sensitive, which occurred at the early stage of differentiation. In the presence of AOPPs, induction of preadipocytes differentiation resulted in upregulated expression of C/EBP homologous protein (CHOP) and CUG-Triplet repeat-binding protein (CUGBP), two important inhibitors of preadipocytes differentiation. In addition, treatment with AOPPs increased abundance of C/EBP-beta-liver enriched inhibitory protein (C/EBP-beta-LIP), a truncated C/EBP-beta isoform without adipogenic activity. Moreover, AOPPs-treated preadipocytes expressed a macrophage marker F4/80 and overexpressed tumor necrosis factor-alpha and interleukin-6 via nuclear factor-kappaB (NF-kappaB)-dependent pathway. However, blocking inflammation with NF-kappaB inhibitor failed to improve AOPPs-induced inhibition of preadipocytes differentiation. These data suggest that accumulation of AOPPs may inhibit differentiation of preadipocytes and activate inflammation in these cells. This information might have implication for understanding the impairment of preadipocytes differentiation and fat inflammation seen in metabolic syndrome.

  8. The expression of pluripotency genes and neuronal markers after neurodifferentiation in fibroblasts co-cultured with human umbilical cord blood mononuclear cells.

    PubMed

    Marinowic, D R; Domingues, M F; Machado, D C; DaCosta, J C

    2015-01-01

    Human umbilical cord blood is an attractive source of stem cells; however, it has a heterogeneous cell population with few mesenchymal stem cells. Cell reprogramming induced by different methodologies can confer pluripotency to differentiated adult cells. The objective of this study was to evaluate the reprogramming of fibroblasts and their subsequent neural differentiation after co-culture with umbilical cord blood mononuclear cells. Cells were obtained from four human umbilical cords. The mononuclear cells were cultured for 7 d and subsequently co-cultured with mouse fibroblast NIH-3T3 cells for 6 d. The pluripotency of the cells was evaluated by RT-PCR using primers specific for pluripotency marker genes. The pluripotency was also confirmed by adipogenic and osteogenic differentiation. Neural differentiation of the reprogrammed cells was evaluated by immunofluorescence. All co-cultured cells showed adipogenic and osteogenic differentiation capacity. After co-cultivation, cells expressed the pluripotency gene KLF4. Statistically significant differences in cell area, diameter, optical density, and fractal dimension were observed by confocal microscopy in the neurally differentiated cells. Contact in the form of co-cultivation of fibroblasts with umbilical cord blood mononuclear fraction for 6 d promoted the reprogramming of these cells, allowing the later induction of neural differentiation.

  9. Induction of mutagenesis and transformation in BALB/c-3T3 clone A31-1 cells by diverse chemical carcinogens

    SciTech Connect

    Lubet, R.A. ); Kouri, R.E.; Curren, R.A.; Putman, D.L.; Schechtman, L.M. )

    1990-01-01

    BALB/c-3T3 cells were employed to examine the genotoxic potential of a variety of known chemical carcinogens. BALB/c-3T3 cells displayed a dose-dependent transformation response to a variety of carcinogens (polycyclic hydrocarbons, methylating agents, ethylating agents, aflatoxin B{sub 1} (AFT{sub 1}), and 4-nitroquinoline-N-oxide (4-NQO)). When the ability of these compounds to induce mutagenesis to resistance to the cardiac glycoside ouabain (OUA{sup R}) was examined, the authors found the short chain alkylating agents to be particularly effective mutagens, causing biologic effects at doses below those necessary to induce a transformation response. In contrast, the polycyclic hydrocarbons which were potent transforming agents were weaker, albeit significant, mutagens for the OUA{sup R} locus in this system, while AFB{sub 1} was quite weak. Further studies were performed with 5-azacytidine (5-AZA) and the nongenotoxic carcinogen cinnamyl anthranilate (CIN). 5-AZA was a potent transforming agent, but failed to cause mutagenesis. CIN similarly caused in vitro transformation. When a series of eight structurally diverse compounds were examined in both the BALB/c-3T3 and C3H10T1/2 mouse fibroblast transformation systems, the BALB/c-3T3 system was shown to be sensitive to a wide variety of potential carcinogens, whereas the C3H10T1/2 system proved routinely sensitive only to the polycyclic hydrocarbons.

  10. Combined effects of 60 Hz electromagnetic field exposure with various stress factors on cellular transformation in NIH3T3 cells.

    PubMed

    Lee, Hae-June; Jin, Yeung Bae; Lee, Jae Seon; Choi, Jong-Il; Lee, Ju-Woon; Myung, Sung Ho; Lee, Yun-Sil

    2012-04-01

    Epidemiological studies have suggested that extremely low-frequency magnetic fields (ELF-MF) are associated with an increased incidence of cancer. Studies using in vitro systems have reported mixed results for the effects of ELF-MF alone, and the World Health Organization (WHO) Research Agenda published in 2007 suggested that high priority research should include an evaluation of the co-carcinogenic effects of ELF-MF exposure using in vitro models. Here, the carcinogenic potential of ELF-MF exposure alone and in combination with various stress factors was investigated in NIH3T3 mouse fibroblasts using an in vitro cellular transformation assay. NIH3T3 cells were exposed to a 60 Hz ELF-MF (1 mT) alone or in combination with ionizing radiation (IR), hydrogen peroxide (H₂O₂), or c-Myc overexpression, and the resulting number of anchorage-independent colonies was counted. A 4 h exposure of NIH3T3 cells to ELF-MF alone produced no cell transformation. Moreover, ELF exposure did not influence the transformation activity of IR, H₂O₂, or activated c-Myc in our in vitro assay system, suggesting that 1 mT ELF-MF did not affect any additive or synergistic transformation activities in combination with stress factors such as IR, H₂O₂, or activated c-Myc in NIH3T3 cells.

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

    SciTech Connect

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

    1987-01-01

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

  12. Differential Expression of Wound Fibrotic Factors between Facial and Trunk Dermal Fibroblasts

    PubMed Central

    Kurita, Masakazu; Okazaki, Mutsumi; Kaminishi-Tanikawa, Akiko; Niikura, Mamoru; Takushima, Akihiko; Harii, Kiyonori

    2012-01-01

    Clinically, wounds on the face tend to heal with less scarring than those on the trunk, but the causes of this difference have not been clarified. Fibroblasts obtained from different parts of the body are known to show different properties. To investigate whether the characteristic properties of facial and trunk wound healing are caused by differences in local fibroblasts, we comparatively analyzed the functional properties of superficial and deep dermal fibroblasts obtained from the facial and trunk skin of seven individuals, with an emphasis on tendency for fibrosis. Proliferation kinetics and mRNA and protein expression of 11 fibrosis-associated factors were investigated. The proliferation kinetics of facial and trunk fibroblasts were identical, but the expression and production levels of profibrotic factors, such as extracellular matrix, transforming growth factor-β1, and connective tissue growth factor mRNA, were lower in facial fibroblasts when compared with trunk fibro-blasts, while the expression of antifibrotic factors, such as collagenase, basic fibroblast growth factor, and hepatocyte growth factor, showed no clear trends. The differences in functional properties of facial and trunk dermal fibroblasts were consistent with the clinical tendencies of healing of facial and trunk wounds. Thus, the differences between facial and trunk scarring are at least partly related to the intrinsic nature of the local dermal fibroblasts. PMID:22260504

  13. Differential expression of wound fibrotic factors between facial and trunk dermal fibroblasts.

    PubMed

    Kurita, Masakazu; Okazaki, Mutsumi; Kaminishi-Tanikawa, Akiko; Niikura, Mamoru; Takushima, Akihiko; Harii, Kiyonori

    2012-01-01

    Clinically, wounds on the face tend to heal with less scarring than those on the trunk, but the causes of this difference have not been clarified. Fibroblasts obtained from different parts of the body are known to show different properties. To investigate whether the characteristic properties of facial and trunk wound healing are caused by differences in local fibroblasts, we comparatively analyzed the functional properties of superficial and deep dermal fibroblasts obtained from the facial and trunk skin of seven individuals, with an emphasis on tendency for fibrosis. Proliferation kinetics and mRNA and protein expression of 11 fibrosis-associated factors were investigated. The proliferation kinetics of facial and trunk fibroblasts were identical, but the expression and production levels of profibrotic factors, such as extracellular matrix, transforming growth factor-β1, and connective tissue growth factor mRNA, were lower in facial fibroblasts when compared with trunk fibroblasts, while the expression of antifibrotic factors, such as collagenase, basic fibroblast growth factor, and hepatocyte growth factor, showed no clear trends. The differences in functional properties of facial and trunk dermal fibroblasts were consistent with the clinical tendencies of healing of facial and trunk wounds. Thus, the differences between facial and trunk scarring are at least partly related to the intrinsic nature of the local dermal fibroblasts.

  14. Cloning and expression of two distinct high-affinity receptors cross-reacting with acidic and basic fibroblast growth factors.

    PubMed Central

    Dionne, C A; Crumley, G; Bellot, F; Kaplow, J M; Searfoss, G; Ruta, M; Burgess, W H; Jaye, M; Schlessinger, J

    1990-01-01

    The fibroblast growth factor (FGF) family consists of at least seven closely related polypeptide mitogens which exert their activities by binding and activation of specific cell surface receptors. Unanswered questions have been whether there are multiple FGF receptors and what factors determine binding specificity and biological response. We report the complete cDNA cloning of two human genes previously designated flg and bek. These genes encode two similar but distinct cell surface receptors comprised of an extracellular domain with three immunoglobulin-like regions, a single transmembrane domain, and a cytoplasmic portion containing a tyrosine kinase domain with a typical kinase insert. The expression of these two cDNAs in transfected NIH 3T3 cells led to the biosynthesis of proteins of 150 kd and 135 kd for flg and bek, respectively. Direct binding experiments with radiolabeled acidic FGF (aFGF) or basic FGF (bFGF), inhibition of binding with native growth factors, and Scatchard analysis of the binding data indicated that bek and flg bind either aFGF or bFGF with dissociation constants of (2-15) x 10(-11) M. The high affinity binding of two distinct growth factors to each of two different receptors represents a unique double redundancy without precedence among polypeptide growth factor-receptor interactions. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:1697263

  15. Liraglutide attenuates the osteoblastic differentiation of MC3T3-E1 cells by modulating AMPK/mTOR signaling

    PubMed Central

    Hu, Xiong-Ke; Yin, Xin-Hua; Zhang, Hong-Qi; Guo, Chao-Feng; Tang, Ming-Xing

    2016-01-01

    Liraglutide, a synthetic analogue of glucagon-like peptide-1, is utilized in the treatment of type 2 diabetes and obesity. Liraglutide has been previously demonstrated to prevent osteoblastic differentiation of human vascular smooth muscle cells, resulting in the slowing of arterial calcification, however, its effect on bone formation remains unclear. The present study investigated the effect of liraglutide on osteoblastic differentiation using Alizarin Red S staining, and examined the molecular mechanisms underlying the regulatory effect by western blot analysis. The present study demonstrated that protein expression levels of phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK) were downregulated in MC3T3-E1 cells during osteoblastic differentiation in commercial osteogenic differentiation medium, whereas protein expression levels of transforming growth factor-β (TGF-β) and phosphorylated mammalian target of rapamycin (p-mTOR) increased. Liraglutide was subsequently demonstrated to dose-dependently attenuate the osteoblastic differentiation of MC3T3-E1 cells, to upregulate p-AMPK, and downregulate p-mTOR and TGF-β protein expression levels. Treatment with an AMPK-specific inhibitor, Compound C, eradicated the effect of liraglutide on osteoblastic differentiation, and p-mTOR and TGF-β downregulation. An mTOR activator, MHY1485, also abolished the inhibitory effect of liraglutide on osteoblastic differentiation, and resulted in p-mTOR and TGF-β downregulation, but did not attenuate the liraglutide-induced increase in p-AMPK protein expression levels. The results of the present study demonstrate that liraglutide attenuates osteoblastic differentiation of MC3T3-E1 cells via modulation of AMPK/mTOR signaling. The present study revealed a novel function of liraglutide, which contributes to the understanding of its pharmacological and physiological effects in clinical settings. PMID:27600753

  16. Cytotoxicity of Titanate-Calcium Complexes to MC3T3 Osteoblast-Like Cells

    PubMed Central

    Drury, Jeanie L.; Moussi, Joelle; Taylor-Pashow, Kathryn M. L.

    2016-01-01

    Monosodium titanates (MST) are a relatively novel form of particulate titanium dioxide that have been proposed for biological use as metal sorbents or delivery agents, most recently calcium (II). In these roles, the toxicity of the titanate or its metal complex is crucial to its biological utility. The aim of this study was to determine the cytotoxicity of MST and MST-calcium complexes with MC3T3 osteoblast-like cells; MST-Ca(II) complexes could be useful to promote bone formation in various hard tissue applications. MC3T3 cells were exposed to native MST or MST-Ca(II) complexes for 24–72 h. A CellTiter-Blue® assay was employed to assess the metabolic activity of the cells. The results showed that MST and MST-Ca(II) suppressed MC3T3 metabolic activity significantly in a dose-, time-, and cell-density-dependent fashion. MST-Ca(II) suppressed MC3T3 metabolism in a statistically identical manner as native MST at all concentrations. We concluded that MST and MST-Ca(II) are significantly cytotoxic to MC3T3 cells through a mechanism yet unknown; this is a potential problem to the biological utility of these complexes. PMID:28044136

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

    SciTech Connect

    Papadopoulos, T.; Pfeifer, U. )

    1987-07-01

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

  18. Collagen Expression in Fibroblasts with a Novel LMNA Mutation

    PubMed Central

    Nguyen, Desiree; Leistritz, Dru F.; Turner, Lesley; MacGregor, David; Ohson, Kamal; Dancey, Paul; Martin, George M.; Oshima, Junko

    2007-01-01

    Laminopathies are a group of genetic disorders caused by LMNA mutations; they include muscular dystrophies, lipodystrophies and progeroid syndromes. We identified a novel heterozygous LMNA mutation, L59R, in a patient with the general appearance of mandibuloacral dysplasia and progeroid features. Examination of the nuclei of dermal fibroblasts revealed the irregular morphology characteristic of LMNA mutant cells. The nuclear morphological abnormalities of LMNA mutant lymphoblastoid cell lines were less prominent compared to those of primary fibroblasts. Since it has been reported that progeroid features are associated with increased extracellular matrix in dermal tissues, we compared a subset of these components in fibroblast cultures from LMNA mutants with those of control fibroblasts. There was no evidence of intracellular accumulation or altered mobility of collagen chains, or altered conversion of procollagen to collagen, suggesting that skin fibroblast-mediated matrix production may not play a significant role in the pathogenesis of this particular laminopathy. PMID:17150192

  19. Collagen expression in fibroblasts with a novel LMNA mutation

    SciTech Connect

    Nguyen, Desiree; Leistritz, Dru F.; Turner, Lesley; MacGregor, David; Ohson, Kamal; Dancey, Paul; Martin, George M.; Oshima, Junko . E-mail: picard@u.washington.edu

    2007-01-19

    Laminopathies are a group of genetic disorders caused by LMNA mutations; they include muscular dystrophies, lipodystrophies, and progeroid syndromes. We identified a novel heterozygous LMNA mutation, L59R, in a patient with the general appearance of mandibuloacral dysplasia and progeroid features. Examination of the nuclei of dermal fibroblasts revealed the irregular morphology characteristic of LMNA mutant cells. The nuclear morphological abnormalities of LMNA mutant lymphoblastoid cell lines were less prominent compared to those of primary fibroblasts. Since it has been reported that progeroid features are associated with increased extracellular matrix in dermal tissues, we compared a subset of these components in fibroblast cultures from LMNA mutants with those of control fibroblasts. There was no evidence of intracellular accumulation or altered mobility of collagen chains, or altered conversion of procollagen to collagen, suggesting that skin fibroblast-mediated matrix production may not play a significant role in the pathogenesis of this particular laminopathy.

  20. 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; 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

  1. Neural cell surface differentiation antigen gp130(RB13-6) induces fibroblasts and glioma cells to express astroglial proteins and invasive properties.

    PubMed

    Deissler, H; Blass-Kampmann, S; Bruyneel, E; Mareel, M; Rajewsky, M F

    1999-04-01

    Transient expression of the differentiation and tumor cell surface antigen gp130(RB13-6) characterizes a subset of rat glial progenitor cells susceptible to ethylnitrosourea-induced neurooncogenesis. gp130(RB13-6) is as a member of an emerging protein family of ecto-phosphodiesterases/nucleotide pyrophosphatases that includes PC-1 and the tumor cell motility factor autotaxin. We have investigated the potential role of gp130(RB13-6) in glial differentiation by transfection of three cell lines of different origin that do not express endogenous gp130(RB13-6) (NIH-3T3 mouse fibroblasts; C6 and BT7Ca rat glioma cells) with the cDNA encoding gp130(RB13-6). The effect of gp130(RB13-6) expression was analyzed in terms of overall cell morphology, the expression of glial cell-specific marker proteins, and invasiveness. Transfectant sublines, consisting of 100% gp130(RB13-6)-positive cells, exhibited an altered, bipolar morphology. Fascicular aggregates of fibroblastoid cells subsequently developed into mesh-like patterns. Contrary to the parental NIH-3T3 and BT7Ca cells, the transfectant cells invaded into collagen type I. As shown by immunofluorescence staining of the transfectant sublines as well as of primary cultures composed of gp130(RB13-6)-positive and -negative cells, expression of gp130(RB13-6) induced coexpression of proteins typical for glial cells and their precursors, i.e., glial fibrillary acidic protein, the low affinity nerve growth factor receptor, and the neural proteins Thy-1, Ran-2, and S-100. In accordance with its expression in the immature rat nervous system, gp130(RB13-6) may thus have a significant role in the glial differentiation program and its subversion in neurooncogenesis.

  2. Expression of fibroblast growth factor 23 by canine soft tissue sarcomas.

    PubMed

    Hardcastle, M R; Dittmer, K E

    2016-09-01

    Tumour-induced osteomalacia (TIO) is a rare paraneoplastic syndrome of humans. Some mesenchymal tumours (often resembling haemangiopericytomas) express molecules that normally regulate phosphorus metabolism; most frequently, fibroblast growth factor 23. Patients develop renal phosphate wasting and inappropriately low serum concentrations of 1, 25 (OH)2 vitamin D3 , leading to osteomalacia. Surgical removal of the tumour is curative. The authors examined expression of canine fibroblast growth factor 23 in 49 soft tissue sarcomas, and control tissues from normal adult dogs. RNA extracted from bone or formalin-fixed, paraffin-embedded tissues was analysed by end point and quantitative reverse transcriptase-polymerase chain reaction. Fibroblast growth factor 23 expression was detected in bone, lung, kidney, lymph node and thymus. Fifteen of 49 sarcomas (31%) expressed fibroblast growth factor 23, three of these had high relative expression and some features resembling phosphatonin-expressing mesenchymal tumours of humans. Further work is required to determine whether TIO may occur in dogs.

  3. Increased KGF expression promotes fibroblast activation in a double paracrine manner resulting in cutaneous fibrosis.

    PubMed

    Canady, Johanna; Arndt, Stephanie; Karrer, Sigrid; Bosserhoff, Anja K

    2013-03-01

    Fibrotic disorders of the skin share the characteristic features of increased production and deposition of extracellular matrix components by activated fibroblasts. Their clinical course ranges from benign with localized cutaneous involvement to a systemic, life-threatening disease. The molecular cause for fibroblast activation remains unknown, yet epithelial-mesenchymal interactions draw mounting attention in the research field of fibrogenesis. We examined keratinocyte growth factor (KGF), a crucial molecule in fibroblast-keratinocyte cross talk, exemplarily in keloid and scleroderma, and found its expression to be increased in disease-derived fibroblasts and tissues compared with healthy controls. This overexpression induces fibroblast activation through a double paracrine mode of action. Upon KGF stimulation, the keratinocytes produced and secreted OSM (oncostatin M). Fibroblasts were in turn activated by OSM reacting with the increased expression of collagen type I-α1, fibroblast activation protein, and enhanced migration. The observed increase in collagen expression and fibroblast migration can be traced back to OSM-regulated STAT3 phosphorylation, leading to enhanced urokinase plasminogen activator expression. Hence, we propose a causative loop in the pathogenesis of fibrosing disorders of the skin mediated by the overexpression of KGF in mesenchymal cells.

  4. Effect of Biodegradable Shape-Memory Polymers on Proliferation of 3T3 Cells

    NASA Astrophysics Data System (ADS)

    Xu, Shuo-Gui; Zhang, Peng; Zhu, Guang-Ming; Jiang, Ying-Ming

    2011-07-01

    This article evaluates the in vitro biocompatibility for biodegradable shape-memory polymers (BSMP) invented by the authors. 3T3 cells (3T3-Swiss albino GNM 9) of primary and passaged cultures were inoculated into two kinds of carriers: the BSMP carrier and the control group carrier. Viability, proliferation, and DNA synthesis (the major biocompatibility parameters), were measured and evaluated for both the BSMP and naked carrier via cell growth curve analysis, MTT colorimetry and addition of 3H-TdR to culture media. The results showed that there was no difference between the BSMP carrier and the control dish in terms of viability, proliferation, and metabolism of the 3T3 cells. Overall, the BSMP carrier provides good biocompatibility and low toxicity to cells in vitro, and could indicate future potential for this medium as a biological material for implants in vivo.

  5. Calcification of MC3T3-E1 cells on titanium and zirconium.

    PubMed

    Umezawa, Takayuki; Chen, Peng; Tsutsumi, Yusuke; Doi, Hisashi; Ashida, Maki; Suzuki, Shoichi; Moriyama, Keiji; Hanawa, Takao

    2015-01-01

    To confirm similarity of hard tissue compatibility between titanium and zirconium, calcification of MC3T3-E1 cells on titanium and zirconium was evaluated in this study. Mirror-polished titanium (Ti) and zirconium (Zr) disks and zirconium-sputter deposited titanium (Zr/Ti) were employed in this study. The surface of specimens were characterized using scanning electron microscopy and X-ray diffraction. Then, the cellular proliferation, differentiation and calcification of MC3T3-E1 cells on specimens were investigated. The surface of Zr/Ti was much smoother and cleaner than those of Ti and Zr. The proliferation of the cell was the same among three specimens, while the differentiation and calcification on Zr/Ti were faster than those on Ti and Zr. Therefore, Ti and Zr showed the identical hard tissue compatibility according to the evaluation with MC3T3-E1 cells. Sputter deposition may improve cytocompatibility.

  6. 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.

  7. Tumor-produced, active Interleukin-1 {beta} regulates gene expression in carcinoma-associated fibroblasts

    SciTech Connect

    Dudas, Jozsef; Fullar, Alexandra; Bitsche, Mario; Schartinger, Volker; Kovalszky, Ilona; Sprinzl, Georg Mathias; Riechelmann, Herbert

    2011-09-10

    Recently we described a co-culture model of periodontal ligament (PDL) fibroblasts and SCC-25 lingual squamous carcinoma cells, which resulted in conversion of normal fibroblasts into carcinoma-associated fibroblasts (CAFs), and in epithelial-mesenchymal transition (EMT) of SCC-25 cells. We have found a constitutive high interleukin-1{beta} (IL1-{beta}) expression in SCC-25 cells in normal and in co-cultured conditions. In our hypothesis a constitutive IL1-{beta} expression in SCC-25 regulates gene expression in fibroblasts during co-culture. Co-cultures were performed between PDL fibroblasts and SCC-25 cells with and without dexamethasone (DEX) treatment; IL1-{beta} processing was investigated in SCC-25 cells, tumor cells and PDL fibroblasts were treated with IL1-{beta}. IL1-{beta} signaling was investigated by western blot and immunocytochemistry. IL1-{beta}-regulated genes were analyzed by real-time qPCR. SCC-25 cells produced 16 kD active IL1-{beta}, its receptor was upregulated in PDL fibroblasts during co-culture, which induced phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1), and nuclear translocalization of NF{kappa}B{alpha}. Several genes, including interferon regulatory factor 1 (IRF1) interleukin-6 (IL-6) and prostaglandin-endoperoxide synthase 2 (COX-2) were induced in CAFs during co-culture. The most enhanced induction was found for IL-6 and COX-2. Treatment of PDL fibroblasts with IL1-{beta} reproduced a time- and dose-dependent upregulation of IL1-receptor, IL-6 and COX-2. A further proof was achieved by DEX inhibition for IL1-{beta}-stimulated IL-6 and COX-2 gene expression. Constitutive expression of IL1-{beta} in the tumor cells leads to IL1-{beta}-stimulated gene expression changes in tumor-associated fibroblasts, which are involved in tumor progression. -- Graphical abstract: SCC-25 cells produce active, processed IL1-{beta}. PDL fibroblasts possess receptor for IL1-{beta}, and its expression is increased 4.56-times in the

  8. Tauroursodeoxycholic acid inhibits TNF-α-induced lipolysis in 3T3-L1 adipocytes via the IRE-JNK-perilipin-A signaling pathway.

    PubMed

    Xia, Wenyan; Zhou, Yu; Wang, Lijing; Wang, Linxi; Liu, Xiaoying; Lin, Yichuan; Zhou, Qing; Huang, Jianqing; Liu, Libin

    2017-04-01

    The present study investigated the effects of tauroursodeoxycholic acid (TUDCA) on the lipolytic action of tumor necrosis factor (TNF)-α in 3T3-L1 adipocytes. Following treatment with TNF‑α, cell viability was determined by MTT assay to select the optimum concentration and duration of TNF‑α treatment in 3T3‑L1 adipocytes. Intracellular lipid droplet dispersion and glycerin content in culture media were determined to evaluate the effect of TUDCA on TNF‑α‑induced lipolysis in 3T3‑L1 adipocytes. Western blotting was performed to detect protein expression levels of perilipin‑A and protein markers of endoplasmic reticulum stress: Immunoglobulin‑binding protein (BiP), inositol‑requiring enzyme (IRE), c‑Jun N‑terminal kinase (JNK), phosphorylated (p)‑IRE and p‑JNK. Following treatment with 50 ng/ml TNF‑α for 24 h, glycerin content increased significantly and lipid droplets were dispersed. Glycerin content was reduced significantly and dispersal of lipid droplets reduced following pretreatment of 3T3‑L1 adipocytes with 1 mmol/l TUDCA. TNF‑α additionally activated the expression of BiP, p‑IRE and p‑JNK in a time‑dependent manner; following pretreatment of 3T3‑L1 adipocytes with 1 mmol/l TUDCA, the expression levels of these three proteins decreased. Therefore, TUDCA may inhibit TNF-α-induced lipolysis in 3T3‑L1 adipocytes and reduce production of free fatty acids. Its underlying molecular mechanisms are potentially associated with the inhibition of activation of the IRE‑JNK signaling pathway, which influences perilipin-A expression levels.

  9. MicroRNA-125b-5p inhibits proliferation and promotes adipogenic differentiation in 3T3-L1 preadipocytes.

    PubMed

    Ouyang, Dan; Ye, Yaqiong; Guo, Dongguang; Yu, Xiaofang; Chen, Jian; Qi, Junjie; Tan, Xiaotong; Zhang, Yuan; Ma, Yongjiang; Li, Yugu

    2015-05-01

    Previous evidence has indicated that the microRNA-125b (miR-125b) family plays important roles in the regulation of cancer cell growth, development, differentiation, and apoptosis. However, whether they contribute to the process of adipocyte differentiation remains unclear. In the present study, we revealed that the expression level of miR-125b-5p, a member of miR-125b family, was dramatically up-regulated during differentiation of 3T3-L1 preadipocyte into mature adipocyte. Supplement of miR-125b-5p into 3T3-L1 cells promoted adipogenic differentiation as evidenced by increased lipid droplets and mRNA levels of adipocyte-specific molecular markers, including peroxisome proliferators-activated receptor γ, CCAAT/enhancer-binding protein α, fatty acid-binding protein 4, and lipoprotein lipase, and by triglyceride accumulation. CCK-8 assay showed that miR-125b-5p supplementation significantly inhibited cell proliferation. Flow cytometry analysis showed that miR-125b-5p impaired G1/S phase transition as well as the mRNA and protein expression of G1/S-related genes, such as Cyclin D2, Cyclin D3, and CDK4. Nevertheless, it had no effect on apoptosis. Additionally, by target gene prediction, we demonstrated that smad4 may be a potential target of miR-125b-5p in mouse 3T3-L1 preadipocytes, accounting for some of miR-125b-5p's functions. Taken together, these data indicated that miR-125b-5p may serve as an important positive regulator in adipocyte differentiation, at least partially through down-regulating smad4.

  10. Cytotoxicity of folic acid conjugated hollow silica nanoparticles toward Caco2 and 3T3 cells, with and without encapsulated DOX.

    PubMed

    Patel, Kunal; Sundara Raj, Behin; Chen, Yan; Lou, Xia

    2016-04-01

    Hollow silica nanoparticles of two sizes with and without a folic acid targeting ligand were synthesized. Fickian diffusion of the antitumor drug doxorubicin hydrochloride (DOX) was demonstrated by the produced nanoparticles, achieving a cumulative release of 73% and 45% for 215 nm and 430 nm particles respectively over a period of 500 h. The hollow silica nanoparticles presented a time and dose dependent toxicity, selective to human epithelial colorectal adenocarcinoma (Caco2) cells, over mouse embryonic fibroblast (3T3) cells. At 24h Caco2 cell viability was reduced to 66% using pure hollow silica at a concentration of 50 μg mL(-1), while that of 3T3 cells remained at 94% under the same conditions. The selective cytotoxicity of hollow silica nanoparticles was further enhanced by conjugation of folic acid and incorporation of DOX: at 24h and an equivalent DOX concentration of 0.5 μg mL(-1), viable Caco2 cells were reduced to 45% while 3T3 cells were reduced to 83%. Interestingly the equivalent dose of free DOX was more toxic to 3T3 than to Caco2 cells, reducing the 3T3 viability to 72% and the Caco2 viability to 80%, which is likely due to the presence of the p-glycoprotein pumps in Caco2 cells. Folic acid conjugation served to enhance the viability of both cell lines in this work. Careful optimization of the folate content should further improve the cell specificity of the hollow silica nanoparticles, thus providing a viable targeting platform for cancer therapy.

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

    SciTech Connect

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

    2009-02-01

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

  12. Phosphoprotein phosphatase 1CB (PPP1CB), a novel adipogenic activator, promotes 3T3-L1 adipogenesis.

    PubMed

    Cho, Young-Lai; Min, Jeong-Ki; Roh, Kyung Min; Kim, Won Kon; Han, Baek Soo; Bae, Kwang-Hee; Lee, Sang Chul; Chung, Sang J; Kang, Hyo Jin

    2015-11-13

    Understanding the molecular networks that regulate adipogenesis is crucial for gaining insight into obesity and identifying medicinal targets thereof is necessary for pharmacological interventions. However, the identity and molecular actions of activators that promote the early development of adipocytes are still largely unknown. Here, we demonstrate a novel role for phosphoprotein phosphatase 1CB (PPP1CB) as a potent adipogenic activator that promotes adipocyte differentiation. PPP1CB expression increased in vitro during the early phase of 3T3-L1 adipogenesis and in the murine model of high-fat diet-induced obesity. Depletion of PPP1CB dramatically suppressed the differentiation of 3T3-L1 cells into mature adipocytes, with a concomitant change in adipocyte marker genes and significantly inhibited clonal expansion. We also showed that knockdown of PPP1CB caused a significant decrease in C/EBPδ expression, which in turn resulted in attenuation of PPARγ, C/EBPα, adiponectin, and aP2. In addition, we elucidated the functional significance of PPP1CB by linking p38 activation to C/EBPδ expression in early adipogenesis. Overall, our findings demonstrate a novel function of PPP1CB in promoting adipogenesis and suggest that PPP1CB may be a promising therapeutic target for treatment of obesity and obesity-related diseases.

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

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  15. Changes in gap junction organization and decreased coupling during induced apoptosis in lens epithelial and NIH-3T3 cells.

    PubMed

    Theiss, Carsten; Mazur, Antonina; Meller, Karl; Mannherz, Hans Georg

    2007-01-01

    We demonstrate that global induction of apoptosis in primary bovine lens epithelial (LEC) or fibroblastic mouse NIH-3T3 cells by staurosporine, puromycin, cycloheximide, or etoposide is accompanied by a decrease in coupling by gap junctions. Cell coupling as tested by neurobiotin spreading was maintained when the LEC or NIH-3T3 cells were pre-incubated with the pan-caspase inhibitor zVAD or the caspase-3 inhibiting tetrapeptide DEVD. Immunohistochemistry using anti-connexin-43 antibodies showed a reduction of plasma membrane integrated connexin-43 in both cell lines when undergoing apoptosis. Western blotting indicated degradation of connexin-43 that was inhibited by zVAD or DEVD. Cell coupling at single cell level was tested by direct microinjecting into LEC apoptosis-inducing agents of low molecular mass like staurosporine, etoposide and puromycin or the high molecular mass proteins caspase-3 and -8 in activated state. Microinjection of puromycin or etoposide induced apoptotic morphological changes of only the injected cell within 90 or 180 min, but did not affect adjacent cells. In contrast, microinjection of staurosporine led to a rapid induction of apoptosis of the injected and a number of adjacent cells suggesting spreading of staurosporine most probably through gap junction pores held open by dephosphorylation of connexin-43 as verified by immunoblotting and staining using a phospho-serine368-specific anti-connexin-43 antibody. Microinjection of active caspase-8 led after 3 h to morphological apoptotic alterations of only the injected cell, but did not inhibit spreading of co-injected neurobiotin to neighboring cells during the first hour. In contrast, microinjection of active caspase-3-induced apoptosis only of the injected cell after 60 min and rapidly and completely suppressed coupling to neighboring cells.

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

    PubMed

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

    2014-05-01

    Quantitative reverse transcription PCR (qRT-PCR) is becoming increasingly important in the effort to gain insight into the molecular mechanisms underlying adipogenesis. However, the expression profile of a target gene may be misinterpreted due to the unstable expression of the reference genes under different experimental conditions. Therefore, in this study, we investigated the expression stability of 10 commonly used reference genes during 3T3-L1 adipocyte differentiation. The mRNA expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and transferrin receptor (TFRC) significantly increased during the course of 3T3-L1 adipocyte differentiation, which was decreased by berberine, an inhibitor of adipogenesis. Three popular algorithms, GeNorm, NormFinder and BestKeeper, identified 18 ribosomal RNA and hydroxymethylbilane synthase (HMBS) as the most stable reference genes, while GAPDH and TFRC were the least stable ones. Peptidylprolyl isomerase A [PIPA (cyclophilin A)], ribosomal protein, large, P0 (36-B4), beta-2-microglobulin (B2M), α1-tubulin, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and β-actin showed relatively stable expression levels. The choice of reference genes with various expression stabilities exerted a profound influence on the expression profiles of 2 target genes, peroxisome proliferator-activated receptor (PPAR)γ2 and C/EBPα. In addition, western blot analysis revealed that the increased protein expression of GAPDH was markedly inhibited by berberine during adipocyte differentiation. This study highlights the importance of selecting suitable reference genes for qRT-PCR studies of gene expression during the process of adipogenesis.

  17. 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 α subunits of Gs (Gαs) and G14 (Gα14) 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 γ (PPARγ and CCAAT/enhancer-binding protein α (C/EBPα 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 Gαs but not YM-254890, an inhibitor of Gα14, 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 Gαs 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

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

    PubMed

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

    2014-02-01

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

  19. 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.

  20. Antiadopogenic effects of rice hull smoke extract in 3T3-L1 cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The present study investigates the inhibitory effects of a rice hull smoke extract (RHSE) against adipogenesis in 3T3-L1 pre-adipocyte cells. At concentrations of 0.1% and 0.5% RHSE, MDI-induced cells were shown to reduce their cellular lipid content by about 72% and 88%, respectively, compared to ...

  1. Interferon Regulatory Factor 4 Contributes to Transformation of v-Rel-Expressing Fibroblasts

    PubMed Central

    Hrdličková, Radmila; Nehyba, Jiří; Bose, Henry R.

    2001-01-01

    The avian homologue of the interferon regulatory factor 4 (IRF-4) and a novel splice variant lacking exon 6, IRF-4ΔE6, were isolated and characterized. Chicken IRF-4 is expressed in lymphoid organs, less in small intestine, and lungs. IRF-4ΔE6 mRNA, though less abundant than full-length IRF-4, was detected in lymphoid tissues, with the highest levels observed in thymic cells. IRF-4 is highly expressed in v-Rel-transformed lymphocytes, and the expression of IRF-4 is increased in v-Rel- and c-Rel-transformed fibroblasts relative to control cells. The expression of IRF-4 from retrovirus vectors morphologically transformed primary fibroblasts, increased their saturation density, proliferation, and life span, and promoted their growth in soft agar. IRF-4 and v-Rel cooperated synergistically to transform fibroblasts. The expression of IRF-4 antisense RNA eliminated formation of soft agar colonies by v-Rel and reduced the proliferation of v-Rel-transformed cells. v-Rel-transformed fibroblasts produced interferon 1 (IFN1), which inhibits fibroblast proliferation. Infection of fibroblasts with retroviruses expressing v-Rel resulted in an increase in the mRNA levels of IFN1, the IFN receptor, STAT1, JAK1, and 2′,5′-oligo(A) synthetase. The exogenous expression of IRF-4 in v-Rel-transformed fibroblasts decreased the production of IFN1 and suppressed the expression of several genes in the IFN transduction pathway. These results suggest that induction of IRF-4 expression by v-Rel likely facilitates transformation of fibroblasts by decreasing the induction of this antiproliferative pathway. PMID:11533227

  2. AGE-RELATED GENE EXPRESSION CHANGES IN HUMAN SKIN FIBROBLASTS INDUCED BY MMS

    EPA Science Inventory

    Age-Related Gene Expression Changes In Human Skin Fibroblasts Induced By methyl methanesulfonate. Geremy W. Knapp, Alan H. Tennant, and Russell D. Owen. Environmental Carcinogenesis Division, National Health and Environmental Effects Research Laboratory, U. S. Environmental Prote...

  3. Hernia fibroblasts lack β-estradiol induced alterations of collagen gene expression

    PubMed Central

    2006-01-01

    Background Estrogens are reported to increase type I and type III collagen deposition and to regulate Metalloproteinase 2 (MMP-2) expression. These proteins are reported to be dysregulated in incisional hernia formation resulting in a significantly decreased type I to III ratio. We aimed to evaluate the β-estradiol mediated regulation of type I and type III collagen genes as well as MMP-2 gene expression in fibroblasts derived from patients with or without history of recurrent incisional hernia disease. We compared primary fibroblast cultures from male/female subjects without/without incisional hernia disease. Results Incisional hernia fibroblasts (IHFs) revealed a decreased type I/III collagen mRNA ratio. Whereas fibroblasts from healthy female donors responded to β-estradiol, type I and type III gene transcription is not affected in fibroblasts from males or affected females. Furthermore β-estradiol had no influence on the impaired type I to III collagen ratio in fibroblasts from recurrent hernia patients. Conclusion Our results suggest that β-estradiol does not restore the imbaired balance of type I/III collagen in incisional hernia fibroblasts. Furthermore, the individual was identified as an independent factor for the β-estradiol induced alterations of collagen gene expression. The observation of gender specific β-estradiol-dependent changes of collagen gene expression in vitro is of significance for future studies of cellular response. PMID:17010202

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

    SciTech Connect

    Inoue, Kazushi; Akiyama, Tetsu; Toyoshima, Kumao ); Wongsasant, Budsaba )

    1991-12-01

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

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

    PubMed

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

    2013-05-01

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

  6. Effects of Various 5,7-Dihydroxyflavone Analogs on Adipogenesis in 3T3-L1 Cells.

    PubMed

    Nishina, Atsuyoshi; Ukiya, Motohiko; Fukatsu, Makoto; Koketsu, Mamoru; Ninomiya, Masayuki; Sato, Daisuke; Yamamoto, Junpei; Kobayashi-Hattori, Kazuo; Okubo, Takeshi; Tokuoka, Hideyo; Kimura, Hirokazu

    2015-01-01

    We studied the effects of twelve 5,7-dihydroxyflavone analogs on adipogenesis in 3T3-L1 cells. Among the compounds, luteolin, diosmetin, and chrysoeriol partly inhibited adipogenesis by blocking the accumulation of triacylglycerol in the cells. Conversely, tricetin facilitated triacylglycerol accumulation in the cells. The induction of lipogenesis or lipolysis may depend on the number and bonding position of hydroxyl or methoxy groups on the B ring of 5,7-dihydroxyflavone. The mRNA expression levels of adipogenic and lipogenic genes were suppressed by luteolin treatment in the cells, while the mRNA levels of lipolytic genes were not affected. However, the expression levels of the adipogenic, lipogenic, and lipolytic genes, except for adipocyte protein 2 (aP2), were not affected by the addition of tricetin. Moreover, luteolin suppressed glucose transporter type 4 (GLUT4) gene and protein levels. These results indicate that luteolin decreased triacylglycerol levels in 3T3-L1 cells during adipogenesis through the suppression of adipogenic/lipogenic and GLUT4 genes and GLUT4 protein.

  7. Soyasaponins Aa and Ab exert an anti-obesity effect in 3T3-L1 adipocytes through downregulation of PPARγ.

    PubMed

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

    2015-02-01

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

  8. Assay to evaluate BAL Fluid regulation of Fibroblast α-SMA Expression

    PubMed Central

    Larson-Casey, Jennifer L.; Carter, A. Brent

    2016-01-01

    Because transforming growth factor-β (TGF-β1) induces differentiation of fibroblasts to myofibroblasts, we developed a protocol to evaluate alveolar macrophage-derived TGF-β1 regulation of lung fibroblast differentiation (Larson-Casey et al., 2016). The protocol allows evaluating the ability of mouse bronchoalveolar lavage (BAL) fluid to alter fibroblast differentiation. Fibroblast differentiation was measured by the expression of α-smooth muscle actin (α-SMA). Background Alveolar macrophages play an integral role in pulmonary fibrosis development by increasing the expression of TGF-β1 (He et al., 2011). Our prior data demonstrate that alveolar macrophages are a critical source of TGF-β1 as mice harboring a conditional deletion of TGF-β1 in macrophages were protected from pulmonary fibrosis (Larson-Casey et al., 2016). The expression of α-SMA is a defining feature of myofibroblasts, and TGF-β1 is a well-characterized pro-fibrotic mediator that induces transformation of fibroblasts to myofibroblasts both in vitro (Desmoulière et al., 1993) and in vivo (Sime et al., 1997). Prior studies exposed fibroblasts to recombinant TGF-β1 to show its effect on differentiation and function (Horowitz et al., 2007). Here we have developed a protocol for determining the ability of mouse BAL fluid to alter the differentiation of human lung fibroblasts to myofibroblasts, the cells that produce extracellular matrix proteins. PMID:28239621

  9. Gene Expression Profiling of IL-17A-Treated Synovial Fibroblasts from the Human Temporomandibular Joint

    PubMed Central

    Hattori, Toshio; Ogura, Naomi; Akutsu, Miwa; Kawashima, Mutsumi; Watanabe, Suguru; Ito, Ko; Kondoh, Toshirou

    2015-01-01

    Synovial fibroblasts contribute to the inflammatory temporomandibular joint under pathogenic stimuli. Synovial fibroblasts and T cells participate in the perpetuation of joint inflammation in a mutual activation feedback, via secretion of cytokines and chemokines that stimulate each other. IL-17 is an inflammatory cytokine produced primarily by Th17 cells which plays critical role in the pathogenesis of numerous autoimmune and inflammatory diseases. Here, we investigated the roles of IL-17A in temporomandibular joint disorders (TMD) using genome-wide analysis of synovial fibroblasts isolated from patients with TMD. IL-17 receptors were expressed in synovial fibroblasts as assessed using real-time PCR. Microarray analysis indicated that IL-17A treatment of synovial fibroblasts upregulated the expression of IL-6 and chemokines. Real-time PCR analysis showed that the gene expression of IL-6, CXCL1, IL-8, and CCL20 was significantly higher in IL-17A-treated synovial fibroblasts compared to nontreated controls. IL-6 protein production was increased by IL-17A in a time- and a dose-dependent manner. Additionally, IL-17A simulated IL-6 protein production in synovial fibroblasts samples isolated from three patients. Furthermore, signal inhibitor experiments indicated that IL-17-mediated induction of IL-6 was transduced via activation of NFκB and phosphatidylinositol 3-kinase/Akt. These results suggest that IL-17A is associated with the inflammatory progression of TMD. PMID:26839464

  10. Alteration of Connective Tissue Growth Factor (CTGF) Expression in Orbital Fibroblasts from Patients with Graves' Ophthalmopathy.

    PubMed

    Tsai, Chieh-Chih; Wu, Shi-Bei; Chang, Pei-Chen; Wei, Yau-Huei

    2015-01-01

    Graves' ophthalmopathy (GO) is a disfiguring and sometimes blinding disease, which is characterized by inflammation and swelling of orbital tissues, with fibrosis and adipogenesis being predominant features. The aim of this study is to investigate whether the expression levels of fibrosis-related genes, especially that of connective tissue growth factor (CTGF), are altered in orbital fibroblasts of patients with GO. The role of oxidative stress in the regulation of CTGF expression in GO orbital fibroblasts is also examined. By a SYBR Green-based real time quantitative PCR (RT-QPCR), we demonstrated that the mRNA expression levels of fibronectin, apolipoprotein J, and CTGF in cultured orbital fibroblasts from patients with GO were significantly higher than those of age-matched normal controls (p = 0.007, 0.037, and 0.002, respectively). In addition, the protein expression levels of fibronectin, apolipoprotein J, and CTGF analyzed by Western blot were also significantly higher in GO orbital fibroblasts (p = 0.046, 0.032, and 0.008, respectively) as compared with the control. Furthermore, after treatment of orbital fibroblasts with a sub-lethal dose of hydrogen peroxide (200 μM H2O2), we found that the H2O2-induced increase of CTGF expression was more pronounced in the GO orbital fibroblasts as compared with those in normal controls (20% vs. 7%, p = 0.007). Importantly, pre-incubation with antioxidants including N-acetylcysteine (NAC) and vitamin C, respectively, resulted in significant attenuation of the induction of CTGF in GO orbital fibroblasts in response to H2O2 (p = 0.004 and 0.015, respectively). Taken together, we suggest that oxidative stress plays a role in the alteration of the expression of CTGF in GO orbital fibroblasts that may contribute to the pathogenesis and progression of GO. Antioxidants may be used in combination with the therapeutic agents for effective treatment of GO.

  11. Alteration of Connective Tissue Growth Factor (CTGF) Expression in Orbital Fibroblasts from Patients with Graves’ Ophthalmopathy

    PubMed Central

    Chang, Pei-Chen; Wei, Yau-Huei

    2015-01-01

    Graves’ ophthalmopathy (GO) is a disfiguring and sometimes blinding disease, which is characterized by inflammation and swelling of orbital tissues, with fibrosis and adipogenesis being predominant features. The aim of this study is to investigate whether the expression levels of fibrosis-related genes, especially that of connective tissue growth factor (CTGF), are altered in orbital fibroblasts of patients with GO. The role of oxidative stress in the regulation of CTGF expression in GO orbital fibroblasts is also examined. By a SYBR Green-based real time quantitative PCR (RT-QPCR), we demonstrated that the mRNA expression levels of fibronectin, apolipoprotein J, and CTGF in cultured orbital fibroblasts from patients with GO were significantly higher than those of age-matched normal controls (p = 0.007, 0.037, and 0.002, respectively). In addition, the protein expression levels of fibronectin, apolipoprotein J, and CTGF analyzed by Western blot were also significantly higher in GO orbital fibroblasts (p = 0.046, 0.032, and 0.008, respectively) as compared with the control. Furthermore, after treatment of orbital fibroblasts with a sub-lethal dose of hydrogen peroxide (200 μM H2O2), we found that the H2O2-induced increase of CTGF expression was more pronounced in the GO orbital fibroblasts as compared with those in normal controls (20% vs. 7%, p = 0.007). Importantly, pre-incubation with antioxidants including N-acetylcysteine (NAC) and vitamin C, respectively, resulted in significant attenuation of the induction of CTGF in GO orbital fibroblasts in response to H2O2 (p = 0.004 and 0.015, respectively). Taken together, we suggest that oxidative stress plays a role in the alteration of the expression of CTGF in GO orbital fibroblasts that may contribute to the pathogenesis and progression of GO. Antioxidants may be used in combination with the therapeutic agents for effective treatment of GO. PMID:26599235

  12. Dehydrocostus lactone prevents mitochondrial dysfunction in osteoblastic MC3T3-E1 cells.

    PubMed

    Choi, Eun Mi

    2011-08-16

    The dried root of Saussurea lappa Clarke (Compositae) has been used as a traditional medicine. Dehydrocostus lactone is one of the main bioactive constituents of this medicinal plant. In the present study, the protective effect of dehydrocostus lactone against antimycin A (an inhibitor of mitochondrial complex III)-induced cytotoxicity was investigated in osteoblastic MC3T3-E1 cells. Pre-treatment with dehydrocostus lactone prior to antimycin A exposure significantly prevented mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, cytochrome c release, intracellular calcium elevation and potassium loss, and reactive oxygen species production induced by antimycin A. These results suggest that dehydrocostus lactone protects osteoblastic MC3T3-E1 cells from antimycin A-induced cell damage through the improved mitochondrial function.

  13. Testosterone stimulates glucose uptake and GLUT4 translocation through LKB1/AMPK signaling in 3T3-L1 adipocytes.

    PubMed

    Mitsuhashi, Kazuteru; Senmaru, Takafumi; Fukuda, Takuya; Yamazaki, Masahiro; Shinomiya, Katsuhiko; Ueno, Morio; Kinoshita, Shigeru; Kitawaki, Jo; Katsuyama, Masato; Tsujikawa, Muneo; Obayashi, Hiroshi; Nakamura, Naoto; Fukui, Michiaki

    2016-01-01

    Decreases in serum testosterone concentrations in aging men are associated with metabolic disorders. Testosterone has been reported to increase GLUT4-dependent glucose uptake in skeletal muscle cells and cardiomyocytes. However, studies on glucose uptake occurring in response to testosterone stimulation in adipocytes are currently not available. This study was designed to determine the effects of testosterone on glucose uptake in adipocytes. Glucose uptake was assessed with 2-[(3)H] deoxyglucose in 3T3-L1 adipocytes. GLUT4 translocation was evaluated in plasma membrane (PM) sheets and PM fractions by immunofluorescence and immunoblotting, respectively. Activation of GLUT4 translocation-related protein kinases, including Akt, AMPK, LKB1, CaMKI, CaMKII, and Cbl was followed by immunoblotting. Expression levels of androgen receptor (AR) mRNA and AR translocation to the PM were assessed by real-time RT-PCR and immunoblotting, respectively. The results showed that both high-dose (100 nM) testosterone and testosterone-BSA increased glucose uptake and GLUT4 translocation to the PM, independently of the intracellular AR. Testosterone and testosterone-BSA stimulated the phosphorylation of AMPK, LKB1, and CaMKII. The knockdown of LKB1 by siRNA attenuated testosterone- and testosterone-BSA-stimulated AMPK phosphorylation and glucose uptake. These results indicate that high-dose testosterone and testosterone-BSA increase GLUT4-dependent glucose uptake in 3T3-L1 adipocytes by inducing the LKB1/AMPK signaling pathway.

  14. Spindlin1, a novel nuclear protein with a role in the transformation of NIH3T3 cells.

    PubMed

    Gao, Yanhong; Yue, Wen; Zhang, Peng; Li, Li; Xie, Xiaoyan; Yuan, Hongfeng; Chen, Lin; Liu, Daqing; Yan, Fang; Pei, Xuetao

    2005-09-23

    spindlin1, a novel human gene recently isolated by our laboratory, is highly homologous to mouse spindlin gene. In this study, we cloned cDNA full-length of this novel gene and send it to GenBank database as spindlin1 (Homo sapiens spindlin1) with Accession No. AF317228. In order to investigate the function of spindlin1, we studied further the subcellular localization of Spindlin1 protein and the effects of spindlin1 overexpression in NIH3T3 cells. The results showed that the fusion protein pEGFP-N1-spindlin1 was located in the nucleus and the C-terminal is correlated with nuclear localization of Spindlin1 protein. NIH3T3 cells which could stably express spindlin1 as a result of RT-PCR analysis compared with the control cells displayed a complete morphological change; made cell growth faster; and increased the percentage of cells in G2/M and S phase. Furthermore, overexpressed spindlin1 cells formed colonies in soft agar in vitro and formed tumors in nude mice. Our findings provide direct evidence that spindlin1 gene may contribute to tumorigenesis.

  15. Sodium acetate decreases phosphorylation of hormone sensitive lipase in isoproterenol-stimulated 3T3-L1 mature adipocytes.

    PubMed

    Aberdein, Nicola; Schweizer, Michael; Ball, Derek

    2014-04-01

    Lipolysis, the process of hydrolysis of stored triacylglycerol into glycerol and non-esterified fatty acids (NEFA), is reported to be reduced by short chain fatty acids (SCFA) but the mechanism of this inhibition is poorly understood. The aim of this study was to measure the phosphorylation at serine residue 563 of hormone sensitive lipase with and without exposure to sodium acetate. Using the 3T3-L1 cell line, we identified that stimulating the cells with isoproterenol increased phosphorylated hormone sensitive lipase (pHSL) expression by 60% compared with the basal state. In the presence of the SCFA acetate in stimulated cells, pHSL decreased by 15% compared with stimulated cells alone. These results were mirrored by the NEFA release from stimulated cells that had significantly decreased in the presence of sodium acetate after 60 min (from 0.53 µmol mg(-1) protein to 0.41 µmol mg(-1) protein, respectively, P = 0.004); and 180 min (1.73 µmol mg(-1) protein to 1.13 µmol mg(-1) protein, P = 0.020); however, treatment had no effect on glycerol release (P = 0.109). In conclusion, exposure to 4 mM acetate reduced the level of phosphorylation of HSL(SER563) in mature 3T3-L1 adipocytes and led to a significant reduction in NEFA release, although glycerol release was not affected.

  16. 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 < 0.05) and the cell proliferation with PNS treatment was found during the whole osteogenic period. Moreover, cellular ALP activity with PNS was increased during 7, 14, and 21 d and cell mineralization with PNS was enhanced in 14 and 21 d. Furthermore, the differentiation markers Col1 and OCN increased in the PNS-treated cells. Our work suggests that PNS may stimulate the osteogenesis process which contains osteoblastic proliferation, differentiation, and mineralization by increasing cellular ALP activity, extracellular matrix mineralization, and osteoblast-associated molecules in the osteoblasts.

  17. Catechin and quercetin attenuate adipose inflammation in fructose-fed rats and in 3T3-L1 adipocytes

    PubMed Central

    Vazquez Prieto, Marcela A.; Bettaieb, Ahmed; Rodriguez Lanzi, Cecilia; Soto, Verónica C.; Perdicaro, Diahann J.; Galmarini, Claudio R.; Haj, Fawaz G.; Miatello, Roberto M.; Oteiza, Patricia I.

    2015-01-01

    Scope This study evaluated the capacity of dietary catechin (C), quercetin (Q) and the combination of both (CQ), to attenuate adipose inflammation triggered by high fructose (HFr) consumption in rats and by tumor necrosis factor alpha (TNFα) in 3T3-L1 adipocytes. Methods and results In rats, HFr consumption for 6 wk caused dyslipidemia, insulin resistance, reduced plasma adiponectin, adiposity, and adipose tissue inflammation. Dietary supplementation with 20 mg/kg/d of C, Q and CQ improved all these parameters. In 3T3-L1 adipocytes, C and Q attenuated TNFα-induced elevated protein carbonyls, increased pro-inflammatory cytokine expression (MCP-1, resistin), and decreased adiponectin. The protective effects of C and Q on adipose inflammation are in part associated with their capacity to: i) decrease the activation of the mitogen activated kinases (MAPKs) JNK and p38; and ii) prevent the downregulation of PPARγ. In summary, C and Q, and to a larger extent the combination of both, attenuated adipose pro-inflammatory signaling cascades and regulated the balance of molecules that improve (adiponectin) or impair (TNFα, MCP-1, resistin) insulin sensitivity. Conclusion Together, these findings suggest that dietary Q and C may have potential benefits in mitigating MetS associated adipose inflammation, oxidative stress, and insulin resistance. PMID:25620282

  18. Benzyl butyl phthalate promotes adipogenesis in 3T3-L1 preadipocytes: A High Content Cellomics and metabolomic analysis.

    PubMed

    Yin, Lei; Yu, Kevin Shengyang; Lu, Kun; Yu, Xiaozhong

    2016-04-01

    Benzyl butyl phthalate (BBP) has been known to induce developmental and reproductive toxicity. However, its association with dysregulation of adipogenesis has been poorly investigated. The present study aimed to examine the effect of BBP on the adipogenesis, and to elucidate the underlying mechanisms using the 3T3-L1 cells. The capacity of BBP to promote adipogenesis was evaluated by multiple staining approaches combined with a High Content Cellomics analysis. The dynamic changes of adipogenic regulatory genes and proteins were examined, and the metabolite profile was identified using GC/MC based metabolomic analysis. The High Content analysis showed BBP in contrast with Bisphenol A (BPA), a known environmental obesogen, increased lipid droplet accumulation in a similar dose-dependent manner. However, the size of the lipid droplets in BBP-treated cells was significantly larger than those in cells treated with BPA. BBP significantly induced mRNA expression of transcriptional factors C/EBPα and PPARγ, their downstream genes, and numerous adipogenic proteins in a dose and time-dependent manner. Furthermore, GC/MC metabolomic analysis revealed that BBP exposure perturbed the metabolic profiles that are associated with glyceroneogenesis and fatty acid synthesis. Altogether, our current study clearly demonstrates that BBP promoted the differentiation of 3T3-L1 through the activation of the adipogenic pathway and metabolic disturbance.

  19. SV40 transformation of Swiss 3T3 cells can cause a stable reduction in the calcium requirement for growth

    PubMed Central

    1984-01-01

    A well-characterized SV40-transformed Swiss 3T3 line, SV101, and its revertants were tested for the ability to grow in reduced Ca++ (0.01 mM). Transformants and revertants did not differ from the parent 3T3 line in their Ca++ requirements. All three classes of cells grew less well in low Ca++ than in regular Ca++ (2.0 mM). SV40 transformants were then selected for the ability to grow in reduced Ca++. This new class of transformants was found to grow in 1% serum, grow in soft agarose, have a reorganized actin cytoskeleton, and express viral T antigens, as well as grow well in low Ca++. One of the selected clones was found to be T antigen-negative, yet was transformed in the serum, anchorage, actin, and Ca++ assays. It is possible that this clone was a spontaneous transformant. However, Southern blot analysis revealed the presence of integrated SV40 DNA. In addition, this analysis revealed the absence of an intact early region fragment, which codes for the viral T antigens. One explanation of this result may be that the mechanism of viral transformation for growth in low Ca++ involves viral- host DNA interactions that may not require a fully functional T antigen. In this case SV40 integration may be acting as a nonspecific cellular mutagen. PMID:6094595

  20. Ultrasound associated uptake of chitosan nanoparticles in MC3T3-E1 cells

    NASA Astrophysics Data System (ADS)

    Wu, Junyi

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

  1. Constitutive phosphorylation of a Rac GAP MgcRacGAP is implicated in v-Src-induced transformation of NIH3T3 cells.

    PubMed

    Doki, Noriko; Kawashima, Toshiyuki; Nomura, Yasushi; Tsuchiya, Akiho; Oneyama, Chitose; Akagi, Tsuyoshi; Nojima, Yoshihisa; Kitamura, Toshio

    2009-09-01

    MgcRacGAP plays critical roles in cell division through regulating Rho family small GTPases. As we previously reported, phosphorylation of MgcRacGAP on serine 387 (S387) is induced by Aurora B kinase at the midbody during cytokinesis, which is a critical step of cytokinesis. Phosphorylation of S387-MgcRacGAP converts it from RacGAP to RhoGAP, leading to completion of cytokinesis. Here we show that MgcRacGAP is prominently phosphorylated on S387 even in the interphase of v-Src-transformed NIH3T3 cells in the cytoplasm, but not in the interphase of parental NIH3T3 or H-RasV12-transformed NIH3T3 cells. Interestingly, levels of phosphorylation on S387 (pS387) correlated with soft agar colony-forming abilities of v-Src-transformed NIH3T3 cells. Expression of a phosphorylation-mimic mutant MgcRacGAP-S387D enhanced colony formation of v-Src-transformed NIH3T3 cells. Surprisingly, a Rac1 inhibitor but not kinase inhibitors including Aurora B kinase inhibitor specifically inhibited phosphorylation of S387-MgcRacGAP in v-Src-transformed NIH3T3 cells, suggesting the v-Src-induced pathological positive feedback mechanisms towards Rac1 activation using pS387-MgcRacGAP. These results indicated the difference in the mechanisms between v-Src- and H-RasV12-induced transformation, and should shed some light on pathological roles of disordered phosphorylation of MgcRacGAP at S387 in v-Src-induced cell transformation.

  2. 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.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-11-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2004-01-01

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

  7. miR-23b targets Smad 3 and ameliorates the LPS-inhibited osteogenic differentiation in preosteoblast MC3T3-E1 cells.

    PubMed

    Liu, Hongzhi; Hao, Wei; Wang, Xin; Su, Hao

    2016-04-01

    Lipopolysaccharide (LPS) has been confirmed to be the main inhibitor in osteogenic differentiation, posing a clinical challenge to bone healing, particularly for trauma followed by endotoxinemia/sepsis. However, the molecular mechanism remains ambiguous. miR-23b, which regulates multiple signaling pathways in inflammation, has been shown to be deregulated by LPS. In this study, we examined the LPS-mediated regulation on the expression of miR-23b and Smad 3 in preosteoblast MC3T3-E1 cells. Then we determined the regulation of miR-23b overexpression on the Smad 3 expression and on the LPS-mediated inhibition of bone morphogenetic protein-2 (BMP-2)-induced osteogenic differentiation. Our results demonstrated that LPS significantly downregulated the expression of miR-23b, while upregulating Smad 3 in MC3T3-E1 cells. However, the transfection with miR-23b mimics markedly downregulated the Smad 3 in both mRNA and protein levels, via the specific binding to the 3'-untranslated region (UTR) of Smad 3. Moreover, though LPS markedly downregulated the BMP-2-induced osteogenic differentiation of MC3T3-E1 cells by inhibiting the expression of alkaline phosphatase (ALP), Osteocalcin (OCN), Osteopontin (OPN) and Runt-related transcription factor 2 (RUNX2). The upregulated miR-23b reversed such downregulation of ALP, OCN, OPN and RUNX2 in the MC3T3-E1 cells which were treated both with LPS and BMP-2. In conclusion, our data indicates that miR-23b ameliorates the LPS-mediated inhibition of BMP-2-induced osteogenic differentiation in MC3T3-E1 cells, implying the protective role of miR-23b in the LPS-mediated inhibition of osteogenic differentiation and bone formation.

  8. Low-Dose Bisphenol-A Impairs Adipogenesis and Generates Dysfunctional 3T3-L1 Adipocytes.

    PubMed

    Ariemma, Fabiana; D'Esposito, Vittoria; Liguoro, Domenico; Oriente, Francesco; Cabaro, Serena; Liotti, Antonietta; Cimmino, Ilaria; Longo, Michele; Beguinot, Francesco; Formisano, Pietro; Valentino, Rossella

    2016-01-01

    Environmental endocrine disruptors (EDCs), including bisphenol-A (BPA), have been recently involved in obesity and diabetes by dysregulating adipose tissue function. Our aim was to examine whether prolonged exposure to low doses of BPA could affect adipogenesis and adipocyte metabolic functions. Therefore, 3T3-L1 pre-adipocytes were cultured for three weeks with BPA 1 nM to mimic human environmental exposure. We evaluated BPA effect on cell proliferation, differentiation, gene expression and adipocyte metabolic function. BPA significantly increased pre-adipocyte proliferation (p<0.01). In 3T3-L1 adipocytes differentiated in the presence of BPA, the expression of Peroxisome proliferator-activated receptor gamma (PPARγ), Fatty Acid Binding Protein 4/Adipocyte Protein 2 (FABP4/AP2) and CCAAT/enhancer binding protein (C/EBPα) was increased by 3.5, 1.5 and 3 folds, respectively. Mature adipocytes also showed a significant increase in lipid accumulation (p<0.05) and alterations of insulin action, with significant reduction in insulin-stimulated glucose utilization (p<0.001). Moreover, in mature adipocytes, mRNA levels of Leptin, interleukin-6 (IL6) and interferon-γ (IFNγ) were significantly increased (p<0.05). In conclusion, BPA prolonged exposure at low doses, consistent with those found in the environment, may affect adipocyte differentiation program, enhancing pre-adipocyte proliferation and anticipating the expression of the master genes involved in lipid/glucose metabolism. The resulting adipocytes are hypertrophic, with impaired insulin signaling, reduced glucose utilization and increased pro-inflammatory cytokine expression. Thus, these data supported the hypothesis that BPA exposure, during critical stages of adipose tissue development, may cause adipocyte metabolic dysfunction and inflammation, thereby increasing the risk of developing obesity-related diseases.

  9. Low-Dose Bisphenol-A Impairs Adipogenesis and Generates Dysfunctional 3T3-L1 Adipocytes

    PubMed Central

    Ariemma, Fabiana; D’Esposito, Vittoria; Liguoro, Domenico; Oriente, Francesco; Cabaro, Serena; Liotti, Antonietta; Cimmino, Ilaria; Longo, Michele; Beguinot, Francesco; Formisano, Pietro; Valentino, Rossella

    2016-01-01

    Environmental endocrine disruptors (EDCs), including bisphenol-A (BPA), have been recently involved in obesity and diabetes by dysregulating adipose tissue function. Our aim was to examine whether prolonged exposure to low doses of BPA could affect adipogenesis and adipocyte metabolic functions. Therefore, 3T3-L1 pre-adipocytes were cultured for three weeks with BPA 1nM to mimic human environmental exposure. We evaluated BPA effect on cell proliferation, differentiation, gene expression and adipocyte metabolic function. BPA significantly increased pre-adipocyte proliferation (p<0.01). In 3T3-L1 adipocytes differentiated in the presence of BPA, the expression of Peroxisome proliferator-activated receptor gamma (PPARγ), Fatty Acid Binding Protein 4/Adipocyte Protein 2 (FABP4/AP2) and CCAAT/enhancer binding protein (C/EBPα) was increased by 3.5, 1.5 and 3 folds, respectively. Mature adipocytes also showed a significant increase in lipid accumulation (p<0.05) and alterations of insulin action, with significant reduction in insulin-stimulated glucose utilization (p<0.001). Moreover, in mature adipocytes, mRNA levels of Leptin, interleukin-6 (IL6) and interferon-γ (IFNγ) were significantly increased (p<0.05). In conclusion, BPA prolonged exposure at low doses, consistent with those found in the environment, may affect adipocyte differentiation program, enhancing pre-adipocyte proliferation and anticipating the expression of the master genes involved in lipid/glucose metabolism. The resulting adipocytes are hypertrophic, with impaired insulin signaling, reduced glucose utilization and increased pro-inflammatory cytokine expression. Thus, these data supported the hypothesis that BPA exposure, during critical stages of adipose tissue development, may cause adipocyte metabolic dysfunction and inflammation, thereby increasing the risk of developing obesity-related diseases. PMID:26942597

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

    PubMed

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

    2015-01-01

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

  11. The human transforming growth factor type alpha coding sequence is not a direct-acting oncogene when overexpressed in NIH 3T3 cells.

    PubMed Central

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

    1987-01-01

    A peptide secreted by some tumor cells in vitro imparts anchorage-independent growth to normal rat kidney (NRK) cells and has been termed transforming growth factor type alpha (TGF-alpha). To directly investigate the transforming properties of this factor, the human sequence coding for TGF-alpha was placed under the control of either a metallothionein promoter or a retroviral long terminal repeat. These constructs failed to induce morphological transformation upon transfection of NIH 3T3 cells, whereas viral oncogenes encoding a truncated form of its cognate receptor, the EGF receptor, or another growth factor, sis/platelet-derived growth factor 2, efficiently induced transformed foci. When NIH 3T3 clonal sublines were selected by transfection of TGF-alpha expression vectors in the presence of a dominant selectable marker, they were shown to secrete large amounts of TGF-alpha into the medium, to have downregulated EGF receptors, and to be inhibited in growth by TGF-alpha monoclonal antibody. These results indicated that secreted TGF-alpha interacts with its receptor at a cell surface location. Single cell-derived TGF-alpha-expressing sublines grew to high saturation density in culture. However, when plated as single cells on contact-inhibited monolayers of NIH 3T3 cells, they failed to form colonies, whereas v-sis- and v-erbB-transfected cells formed transformed colonies under the same conditions. Moreover, TGF-alpha-expressing sublines were not tumorigenic in nude mice. These and other results imply that TGF-alpha exerts a growth-promoting effect on the entire NIH 3T3 cell population after secretion into the medium but little, if any, effect on the individual cell synthesizing this factor. It is concluded that the normal coding sequence for TGF-alpha is not a direct-acting oncogene when overexpressed in NIH 3T3 cells. Images PMID:3035551

  12. St. John's wort promotes adipocyte differentiation and modulates NF-κB activation in 3T3-L1 cells.

    PubMed

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

    2014-01-01

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

  13. 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 PPARγ, C/EBPα, 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 PPARγ-the key transcription factor of adipogenesis in pre-adipocytes during their early differentiation processes. PMID:24884680

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

    PubMed Central

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

    2007-01-01

    Background Adiponectin is a key mediator of the metabolic syndrome that is caused by visceral fat accumulation. Adiponectin and its receptors are known to be expressed in osteoblasts, but their actions with regard to bone metabolism are still unclear. In this study, we investigated the effects of adiponectin on the proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells. Results Adiponectin receptor type 1 (AdipoR1) mRNA was detected in the cells by RT-PCR. The adenosine monophosphate-activated protein kinase (AMP kinase) was phosphorylated by both adiponectin and a pharmacological AMP kinase activator, 5-amino-imidazole-4-carboxamide-riboside (AICAR), in the cells. AdipoR1 small interfering RNA (siRNA) transfection potently knocked down the receptor mRNA, and the effect of this knockdown persisted for as long as 10 days after the transfection. The transfected cells showed decreased expressions of type I collagen and osteocalcin mRNA, as determined by real-time PCR, and reduced ALP activity and mineralization, as determined by von Kossa and Alizarin red stainings. In contrast, AMP kinase activation by AICAR (0.01–0.5 mM) in wild-type MC3T3-E1 cells augmented their proliferation, differentiation, and mineralization. BrdU assay showed that the addition of adiponectin (0.01–1.0 μg/ml) also promoted their proliferation. Osterix, but not Runx-2, appeared to be involved in these processes because AdipoR1 siRNA transfection and AICAR treatments suppressed and enhanced osterix mRNA expression, respectively. Conclusion Taken together, this study suggests that adiponectin stimulates the proliferation, differentiation, and mineralization of osteoblasts via the AdipoR1 and AMP kinase signaling pathways in autocrine and/or paracrine fashions. PMID:18047638

  15. Individual Differences in the Expression of Conditioned Fear Are Associated with Endogenous Fibroblast Growth Factor 2

    ERIC Educational Resources Information Center

    Graham, Bronwyn M.; Richardson, Rick

    2016-01-01

    These experiments examined the relationship between the neurotrophic factor fibroblast growth factor 2 (FGF2) and individual differences in the expression of conditioned fear. Experiments 1 and 2 demonstrated that rats naturally expressing low levels of contextual or cued fear have higher levels of hippocampal FGF2 relative to rats that express…

  16. Reactive oxygen species regulatory mechanisms associated with rapid response of MC3T3-E1 cells for vibration stress.

    PubMed

    Zhang, Ling; Gan, Xueqi; Zhu, Zhuoli; Yang, Yang; He, Yuting; Yu, Haiyang

    2016-02-12

    Although many previous studies have shown that refractory period-dependent memory effect of vibration stress is anabolic for skeletal homeostasis, little is known about the rapid response of osteoblasts simply derived from vibration itself. In view of the potential role of reactive oxygen species (ROS) in mediating differentiated activity of osteoblasts, whether and how ROS regulates the rapid effect of vibration deserve to be demonstrated. Our findings indicated that MC3T3-E1 cells underwent decreased gene expression of Runx2, Col-I and ALP and impaired ALP activity accompanied by increased mitochondrial fission immediately after vibration loading. Moreover, we also revealed the involvement of ERK-Drp1 signal transduction in ROS regulatory mechanisms responsible for the rapid effect of vibration stress.

  17. Ubiquitin Ligase NEDD4 Regulates PPARγ Stability and Adipocyte Differentiation in 3T3-L1 Cells

    PubMed Central

    Li, Jing Jing; Wang, Ruishan; Lama, Rati; Wang, Xinjiang; Floyd, Z. Elizabeth; Park, Edwards A.; Liao, Francesca-Fang

    2016-01-01

    Peroxisome proliferator–activated receptor-γ (PPARγ) is a ligand-activated nuclear receptor which controls lipid and glucose metabolism. It is also the master regulator of adipogenesis. In adipocytes, ligand-dependent PPARγ activation is associated with proteasomal degradation; therefore, regulation of PPARγ degradation may modulate its transcriptional activity. Here, we show that neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4), an E3 ubiquitin ligase, interacts with the hinge and ligand binding domains of PPARγ and is a bona fide E3 ligase for PPARγ. NEDD4 increases PPARγ stability through the inhibition of its proteasomal degradation. Knockdown of NEDD4 in 3T3-L1 adipocytes reduces PPARγ protein levels and suppresses adipocyte conversion. PPARγ correlates positively with NEDD4 in obese adipose tissue. Together, these findings support NEDD4 as a novel regulator of adipogenesis by modulating the stability of PPARγ. PMID:27917940

  18. Lentiviral Engineered Fibroblasts Expressing Codon-Optimized COL7A1 Restore Anchoring Fibrils in RDEB

    PubMed Central

    Georgiadis, Christos; Syed, Farhatullah; Petrova, Anastasia; Abdul-Wahab, Alya; Lwin, Su M.; Farzaneh, Farzin; Chan, Lucas; Ghani, Sumera; Fleck, Roland A.; Glover, Leanne; McMillan, James R.; Chen, Mei; Thrasher, Adrian J.; McGrath, John A.; Di, Wei-Li; Qasim, Waseem

    2016-01-01

    Cells therapies, engineered to secrete replacement proteins, are being developed to ameliorate otherwise debilitating diseases. Recessive dystrophic epidermolysis bullosa (RDEB) is caused by defects of type VII collagen, a protein essential for anchoring fibril formation at the dermal-epidermal junction. Whereas allogeneic fibroblasts injected directly into the dermis can mediate transient disease modulation, autologous gene-modified fibroblasts should evade immunological rejection and support sustained delivery of type VII collagen at the dermal-epidermal junction. We demonstrate the feasibility of such an approach using a therapeutic grade, self-inactivating-lentiviral vector, encoding codon-optimized COL7A1, to transduce RDEB fibroblasts under conditions suitable for clinical application. Expression and secretion of type VII collagen was confirmed with transduced cells exhibiting supranormal levels of protein expression, and ex vivo migration of fibroblasts was restored in functional assays. Gene-modified RDEB fibroblasts also deposited type VII collagen at the dermal-epidermal junction of human RDEB skin xenografts placed on NOD-scid IL2Rgammanull recipients, with reconstruction of human epidermal structure and regeneration of anchoring fibrils at the dermal-epidermal junction. Fibroblast-mediated restoration of protein and structural defects in this RDEB model strongly supports proposed therapeutic applications in man. PMID:26763448

  19. Paracrine effects of uterine leucocytes on gene expression of human uterine stromal fibroblasts.

    PubMed

    Germeyer, Ariane; Sharkey, Andrew Mark; Prasadajudio, Mirari; Sherwin, Robert; Moffett, Ashley; Bieback, Karen; Clausmeyer, Susanne; Masters, Leanne; Popovici, Roxana Maria; Hess, Alexandra Petra; Strowitzki, Thomas; von Wolff, Michael

    2009-01-01

    The endometrium contains a distinct population of immune cells that undergo cyclic changes during the menstrual cycle and implantation. The majority of these leucocytes are uterine NK (uNK) cells, however how these cells interact with uterine stromal fibroblasts remains unclear. We therefore investigated the paracrine effect of medium conditioned by uterine decidual leucocytes (which are enriched for uNK cells) on the gene expression profile of endometrial stromal fibroblasts in vitro using a cDNA microarray. Our results, verified by real-time PCR, ELISA and FACS analysis, reveal that soluble factors from uterine leucocytes substantially alter endometrial stromal fibroblast gene expression. The largest group of up-regulated genes found was chemokines and cytokines. These include IL-8, CCL8 and CXCL1, which have also been shown to be stimulated by contact of stromal fibroblasts with trophoblast, suggesting that uNK cells work synergistically to support trophoblast migration during implantation. The decidual leucocytes also up-regulated IL-15 and IL-15Ralpha in stromal fibroblasts which could produce a niche for uNK cells allowing proliferation within and recruitment into the uterus, as seen in bone marrow. Overall this study demonstrates, for the first time, the paracrine communication between uterine leucocytes and uterine stromal fibroblasts, and adds to the understanding of how the uterine immune system contributes to the changes seen within the cycling endometrium.

  20. Pentadecapeptide BPC 157 enhances the growth hormone receptor expression in tendon fibroblasts.

    PubMed

    Chang, Chung-Hsun; Tsai, Wen-Chung; Hsu, Ya-Hui; Pang, Jong-Hwei Su

    2014-11-19

    BPC 157, a pentadecapeptide derived from human gastric juice, has been demonstrated to promote the healing of different tissues, including skin, muscle, bone, ligament and tendon in many animal studies. However, the underlying mechanism has not been fully clarified. The present study aimed to explore the effect of BPC 157 on tendon fibroblasts isolated from Achilles tendon of male Sprague-Dawley rat. From the result of cDNA microarray analysis, growth hormone receptor was revealed as one of the most abundantly up-regulated genes in tendon fibroblasts by BPC 157. BPC 157 dose- and time-dependently increased the expression of growth hormone receptor in tendon fibroblasts at both the mRNA and protein levels as measured by RT/real-time PCR and Western blot, respectively. The addition of growth hormone to BPC 157-treated tendon fibroblasts dose- and time-dependently increased the cell proliferation as determined by MTT assay and PCNA expression by RT/real-time PCR. Janus kinase 2, the downstream signal pathway of growth hormone receptor, was activated time-dependently by stimulating the BPC 157-treated tendon fibroblasts with growth hormone. In conclusion, the BPC 157-induced increase of growth hormone receptor in tendon fibroblasts may potentiate the proliferation-promoting effect of growth hormone and contribute to the healing of tendon.

  1. Tea catechins modulate the glucose transport system in 3T3-L1 adipocytes.

    PubMed

    Ueda, Manabu; Furuyashiki, Takashi; Yamada, Kayo; Aoki, Yukiko; Sakane, Iwao; Fukuda, Itsuko; Yoshida, Ken-Ichi; Ashida, Hitoshi

    2010-11-01

    In this study, we investigated the effects of tea catechins on the translocation of glucose transporter (GLUT) 4 in 3T3-L1 adipocytes. We found that the ethyl acetate fraction of green tea extract, containing abundant catechins, most decreased insulin-induced glucose uptake activity in 3T3-L1 cells. When the cells were treated with 50 μM catechins in the absence or presence of insulin for 30 min, nongallate-type catechins increased glucose uptake activity without insulin, whereas gallate-type catechins decreased insulin-induced glucose uptake activity. (-)-Epicatechin (EC) and (-)-epigallocatechin (EGC), nongallate-type catechins, increased glucose uptake activity in the dose- and time-dependent manner, whereas (-)-catechin 3-gallate (Cg) and (-)-epigallocatechin 3-gallate (EGCg), gallate-type catechins, decreased insulin-induced glucose uptake activity in the dose- and time-dependent manner. When the cells were treated with 50 μM catechins for 30 min, EC and EGC promoted GLUT4 translocation, whereas Cg and EGCg decreased the insulin-induced translocation in the cells. EC and EGC increased phosphorylation of PKCλ/ζ without phosphorylation of insulin receptor (IR) and Akt. Wortmannin and LY294002, inhibitors for phosphatidylinositol 3'-kinase (PI3K), decreased EC- and EGC-induced glucose uptake activity in the cells. Cg and EGCg decreased phosphorylation of PKCλ/ζ in the presence of insulin without affecting insulin-induced phosphorylation of IR, and Akt. Therefore, EC and EGC promote the translocation of GLUT4 through activation of PI3K, and Cg and EGCg inhibit insulin-induced translocation of GLUT4 by the insulin signaling pathway in 3T3-L1 cells.

  2. Evaluation of chylomicron effect on ASP production in 3T3-L1 adipocytes.

    PubMed

    Gao, Ying; Gauvreau, Danny; Cui, Wei; Lapointe, Marc; Paglialunga, Sabina; Cianflone, Katherine

    2011-02-01

    In the past few years, there has been increasing interest in the production and physiological role of acylation-stimulating protein (ASP), identical to C3adesArg, a product of the alternative complement pathway generated through C3 cleavage. Recent studies in C3 (-/-) mice that are ASP deficient have demonstrated a role for ASP in postprandial triglyceride clearance and fat storage. The aim of the present study was to establish a cell model and sensitive ELISA assay for the evaluation of ASP production using 3T3-L1 adipocytes. 3T3-L1 preadipocytes were differentiated into adipocytes, then cultured in different media such as serum-free (SF), Dulbecco's modified Eagle's medium (DMEM)/F12 + 10% fetal calf serum (FBS), and at varying concentrations of chylomicrons and insulin + chylomicrons up to 48 h. ASP production in SF and DMEM/F12 + 10% FBS was compared. Chylomicrons stimulated ASP production in a concentration- and time-dependent manner. By contrast, chylomicron treatment had no effect on the production of C3, the precursor protein of ASP, which was constant over 48 h. Addition of insulin (100 nM) to a low-dose of chylomicrons (100 µg TG/ml) significantly increased ASP production compared with chylomicrons alone at 48 h (P < 0.001). Furthermore, addition of insulin significantly increased C3 secretion at both 18 and 48 h of incubation (P < 0.05, P < 0.001, respectively). Overall, the proportion of ASP to C3 remained constant, indicating no change in the ratio of C3 cleaved to generate ASP. This study demonstrated that 3T3-L1 adipocyte is a useful model for the evaluation of C3 secretion and ASP production by using a sensitive mouse-specific ELISA assay. The stimulation of ASP production with chylomicrons demonstrates a physiologically relevant response, and provides a strategy for further studies on ASP production and function.

  3. Isoliquiritigenin impairs insulin signaling and adipocyte differentiation through the inhibition of protein-tyrosine phosphatase 1B oxidation in 3T3-L1 preadipocytes.

    PubMed

    Park, Sun-Ji; Choe, Young-Geun; Kim, Jung-Hak; Chang, Kyu-Tae; Lee, Hyun-Shik; Lee, Dong-Seok

    2016-07-01

    Isoliquritigenin (ISL) is an abundant dietary flavonoid with a chalcone structure, which is an important constituent in Glycyrrhizae Radix (GR). ISL exhibits anti-oxidant activity, and this activity has been shown to play a beneficial role in various health conditions. However, it is unclear whether the anti-oxidant activity of ISL affects insulin signaling pathway and lipid accumulation of adipocytes. We sought to investigate the effects and molecular mechanisms of ISL on insulin-stimulated adipogenesis in 3T3-L1 cells. We investigated whether ISL attenuates insulin-induced Reactive Oxygen Species (ROS) generation, and whether ISL inhibits the lipid accumulation and the expression of adipogenic-genes during the differentiation of 3T3-L1 cells. ISL blocked the ROS generation, suppressed the lipid accumulation and the expression of adipocyte-specific proteins, which are increased in response to insulin stimulation during adipocyte differentiation of 3T3-L1 cells. We also investigated whether the anti-oxidant capacity of ISL is involved in regulating the molecular events of insulin-signaling cascade in 3T3-L1 adipocytes. ISL restores PTP1B activity by inhibiting PTP1B oxidation and IR/PI3K/AKT phosphorylation during the early stages of insulin-induced adipogenesis. Our findings show that the anti-oxidant capacity of ISL attenuated insulin IR/PI3K/AKT signaling through inhibition of PTP1B oxidation, and ultimately attenuated insulin-induced adipocyte differentiation of 3T3-L1 cells.

  4. In vitro mineralization of MC3T3-E1 osteoblast-like cells on collagen/nano-hydroxyapatite scaffolds coated carbon/carbon composites.

    PubMed

    Cao, Sheng; Li, Hejun; Li, Kezhi; Lu, Jinhua; Zhang, Leilei

    2016-02-01

    Collagen/nano-hydroxyapatite (collagen/nHA) scaffolds were successfully prepared on carbon/carbon composites as bioactive films using the layer-by-layer coating method. Surface characterizations of collagen/nHA scaffolds were detected by scanning electron microscope (SEM), X-ray diffraction (XRD), and Fourier transform infrared (FTIR) spectroscopy. Compressive strengths of the scaffolds were evaluated by a universal test machine. In vitro biological performances were determined using scaffolds seeded with MC3T3-E1 osteoblasts-like cells and cultured in mineralization medium for up to 21 days. In addition, cellular morphologies and several related gene expressions of MC3T3-E1 cells in the scaffolds were also evaluated. Chemical and morphological analysis showed that the scaffolds had uniform pore sizes and unified phase composition. Mechanical testing indicated that the collagen/nHA scaffolds had the highest compressive strength in 50% of strain condition when the proportion of collagen and nano-hydroxyapatite was 1:3. Cellular morphology observations and cytology tests indicated that MC3T3-E1 cells were adhered on these scaffolds and proliferated. SEM photographs and gene expressions showed that mineralized MC3T3-E1 cells and newly formed extra cellular matrix (ECM) filled up the pores of the scaffolds after the 3-week mineralization inducement. Nano-sized apatite particles were secreted from MC3T3-E1 cells and combined with the reconstructed ECM. Collectively, collagen/nHA scaffolds provided C/C composites with a biomimetic surface for cell adhesion, proliferation and mineralized extra cellular matrices formation.

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

    PubMed

    Love, Joseph D; Suzuki, Takashi; 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.

  6. 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

  7. Neuropeptide Y1 Receptor Regulates Glucocorticoid-Induced Inhibition of Osteoblast Differentiation in Murine MC3T3-E1 Cells via ERK Signaling.

    PubMed

    Yu, Wei; Zhu, Chao; Xu, Wenning; Jiang, Leisheng; Jiang, Shengdan

    2016-12-21

    High dose glucocorticoid (GC) administration impairs the viability and function of osteoblasts, thus causing osteoporosis and osteonecrosis. Neuropeptide Y1 receptor (Y1 receptor) is expressed in bone tissues and cells, and regulates bone remodeling. However, the role of Y1 receptor in glucocorticoid-induced inhibition of osteoblast differentiation remains unknown. In the present study, osteoblastic cell line MC3T3-E1 cultured in osteogenic differentiation medium was treated with or without of 10(-7) M dexamethasone (Dex), Y1 receptor shRNA interference, Y1 receptor agonist [Leu(31), Pro(34)]-NPY, and antagonist BIBP3226. Cell proliferation and apoptosis were assessed by cell counting kit-8 (CCK-8) assay and cleaved caspase expression, respectively. Osteoblast differentiation was evaluated by Alizarin Red S staining and osteogenic marker gene expressions. Protein expression was detected by Western blot analysis. Dex upregulated the expression of Y1 receptor in MC3T3-E1 cells associated with reduced osteogenic gene expressions and mineralization. Blockade of Y1 receptor by shRNA transfection and BIBP3226 significantly attenuated the inhibitory effects of Dex on osteoblastic activity. Y1 receptor signaling modulated the activation of extracellular signal-regulated kinases (ERK) as well as the expressions of osteogenic genes. Y1 receptor agonist inhibited ERK phosphorylation and osteoblast differentiation, while Y1 receptor blockade exhibited the opposite effects. Activation of ERK signaling by constitutive active mutant of MEK1 (caMEK) abolished Y1 receptor-mediated Dex inhibition of osteoblast differentiation in MC3T3-E1 cells. Taken together, Y1 receptor regulates Dex-induced inhibition of osteoblast differentiation in murine MC3T3-E1 cells via ERK signaling. This study provides a novel role of Y1 receptor in the process of GC-induced suppression in osteoblast survival and differentiation.

  8. 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/EBPα and PPARγ independent of C/EBPβ 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 PPARγ expression seems to stem from its ability to inhibit Akt and augment the TNFα pathway. The Akt-TSC2-mTORC1 pathway has recently been described for PPARγ expression in adipocytes. In our experiments, TNFα 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)α(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 PPARγ-regulated adipogenesis pathway at all stages and by augmenting TNFα-induced lipolysis and apoptosis in mature adipocytes.

  9. 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/EBPα and PPARγ independent of C/EBPβ 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 PPARγ expression seems to stem from its ability to inhibit Akt and augment the TNFα pathway. The Akt–TSC2–mTORC1 pathway has recently been described for PPARγ expression in adipocytes. In our experiments, TNFα 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 Giα1, 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 PPARγ-regulated adipogenesis pathway at all stages and by augmenting TNFα-induced lipolysis and apoptosis in mature adipocytes. PMID:22292054

  10. 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.

  11. 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.

  12. JMV641: a potent bombesin receptor antagonist that inhibits Swiss 3T3 cell proliferation.

    PubMed

    Azay, J; Gagne, D; Devin, C; Llinares, M; Fehrentz, J A; Martinez, J

    1996-08-27

    The peptides of the bombesin family are involved in stimulation of mitogenesis in various cell lines, including cancerous cell lines. Bombesin receptor antagonists are of great interest to inhibit this proliferation. We have synthesized a potent bombesin receptor antagonist, e.g., compound JMV641 [H-DPhe-Gln-Trp-Ala-Val-Gly-His-NH-*CH[CH2-CH(CH3)2]-**CHOH- (CH2)3-CH3 [*(S); **92% of (S) isomer], in which a pseudopeptide bond mimicking the transition state analogue replaced the peptide bond between the two C-terminal residues. This compound was highly potent to dose-dependently inhibit binding of 125I-GRP to Swiss 3T3 cells (IC50 = 0.85 +/- 0.15 nM) and bombesin-stimulated Swiss 3T3 proliferation (pA2 = 8.78). However, compound JMV641 can inhibit bombesin-induced AP-1 regulated genes that are nuclear messengers mediating the actions of signal transduction pathways stimulated by growth factors.

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

    PubMed

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

    2010-08-01

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

  14. Simvastatin enhances induction of inducible nitric oxide synthase in 3T3-L1 adipocytes.

    PubMed

    Araki, Shunsuke; Dobashi, Kazushige; Asayama, Kohtaro; Shirahata, Akira

    2007-09-01

    The present study was designed to determine whether hydroxymethylglutaryl-CoA reductase inhibitors (statins) modulate the NO production via iNOS in adipocytes stimulated by lipopolysaccharide (L) and tumour necrosis factor-alpha (T). Well-differentiated 3T3-L1 adipocytes significantly produced NO by LT-treatment. Pre-incubation with simvastatin, a lipophilic statin, pravastatin, a hydrophilic one, or Y27632, an inhibitor of Rho kinase, further enhanced the production of NO. The effect of simvastatin was offset by mevalonate and geranylgeranyl pyrophosphate (GGPP) but not by squalene. The mRNA level for iNOS parallelled the NO production. The NF-kappaB was activated by the LT-treatment and was further enhanced by simvastatin, pravastatin or Y27632 addition. Mevalonate and GGPP completely offset the effect of simvastatin. Statins and Y27632 also further increased the interleukin-6 secretion in the LT-treated 3T3-L1 adipocytes. These results suggest that statins, especially lipophilic type, enhance induction of iNOS by inhibiting the small GTP-binding protein signal in adipocytes.

  15. Fibroblast activation protein is expressed by rheumatoid myofibroblast-like synoviocytes

    PubMed Central

    Bauer, Stefan; Jendro, Michael C; Wadle, Andreas; Kleber, Sascha; Stenner, Frank; Dinser, Robert; Reich, Anja; Faccin, Erica; Gödde, Stefan; Dinges, Harald; Müller-Ladner, Ulf; Renner, Christoph

    2006-01-01

    Fibroblast activation protein (FAP), as described so far, is a type II cell surface serine protease expressed by fibroblastic cells in areas of active tissue remodelling such as tumour stroma or healing wounds. We investigated the expression of FAP by fibroblast-like synoviocytes (FLSs) and compared the synovial expression pattern in rheumatoid arthritis (RA) and osteoarthritis (OA) patients. Synovial tissue from diseased joints of 20 patients, 10 patients with refractory RA and 10 patients with end-stage OA, was collected during routine surgery. As a result, FLSs from intensively inflamed synovial tissues of refractory RA expressed FAP at high density. Moreover, FAP expression was co-localised with matrix metalloproteinases (MMP-1 and MMP-13) and CD44 splice variants v3 and v7/8 known to play a major role in the concert of extracellular matrix degradation. The pattern of signals appeared to constitute a characteristic feature of FLSs involved in rheumatoid arthritic joint-destructive processes. These FAP-expressing FLSs with a phenotype of smooth muscle actin-positive myofibroblasts were located in the lining layer of the synovium and differ distinctly from Thy-1-expressing and non-proliferating fibroblasts of the articular matrix. The intensity of FAP-specific staining in synovial tissue from patients with RA was found to be different when compared with end-stage OA. Because expression of FAP by RA FLSs has not been described before, the findings of this study highlight a novel element in cartilage and bone destruction of arthritic joints. Moreover, the specific expression pattern qualifies FAP as a therapeutic target for inhibiting the destructive potential of fibroblast-like synovial cells. PMID:17105646

  16. Increased gene expression of Alzheimer disease beta-amyloid precursor protein in senescent cultured fibroblasts.

    PubMed

    Adler, M J; Coronel, C; Shelton, E; Seegmiller, J E; Dewji, N N

    1991-01-01

    The pathological hallmark of Alzheimer disease is the accumulation of neurofibrillary tangles and neuritic plaques in the brains of patients. Plaque cores contain a 4- to 5-kDa amyloid beta-protein fragment which is also found in the cerebral blood vessels of affected individuals. Since amyloid deposition in the brain increases with age even in normal people, we sought to establish whether the disease state bears a direct relationship with normal aging processes. As a model for biological aging, the process of cellular senescence in vitro was used. mRNA levels of beta-amyloid precursor protein associated with Alzheimer disease were compared in human fibroblasts in culture at early passage and when the same fibroblasts were grown to senescence after more than 52 population doublings. A dramatic increase in mRNA was observed in senescent IMR-90 fibroblasts compared with early-passage cells. Hybridization of mRNA from senescent and early proliferating fibroblasts with oligonucleotide probes specific for the three alternatively spliced transcripts of the gene gave similar results, indicating an increase during senescence of all three forms. A similar, though more modest, increase in message levels was also observed in early-passage fibroblasts made quiescent by serum deprivation; with repletion of serum, however, the expression returned to previous low levels. ELISAs were performed on cell extracts from senescent, early proliferating, and quiescent fibroblasts, and quiescent fibroblasts repleted with serum for over 48 hr, using polyclonal antibodies to a synthetic peptide of the beta-amyloid precursor. The results confirmed that the differences in mRNA expression were partially reflected at the protein level. Regulated expression of beta-amyloid precursor protein may be an important determinant of growth and metabolic responses to serum and growth factors under physiological as well as pathological conditions.

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

    PubMed

    Kowalska, Katarzyna; Olejnik, Anna; Rychlik, Joanna; Grajek, Włodzimierz

    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 (PPARγ, C/EBPα, 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.

  18. P4H9-detected molecule expression on spindle-shaped fibroblasts indicates malignant phenotype of colorectal cancer

    PubMed Central

    Yokoyama, Shozo; Ieda, Junji; Yamamoto, Naoyuki; Yamaguchi, Shunsuke; Mitani, Yasuyuki; Takeuchi, Akihiro; Takifuji, Katsunari; Hotta, Tsukasa; Matsuda, Kenji; Watanabe, Takashi; Shively, John E; Yamaue, Hiroki

    2015-01-01

    Background: Our previous study using a mammary fat pad mouse model showed that P4H9, produced by the β2 integrin epitope, detected a molecule on fibroblasts in response to carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1)-expressing cancer cells. P4H9-detected molecule (PDM) expression appeared to be associated with myofibroblast differentiation. In this study, we investigated whether PDM is expressed on fibroblasts and cancer cells in clinical tissue samples, and whether the presence of PDM-expressing colorectal cancer cells is correlated with clinicopathological features of patients. Methods: Immunohistochemistry was conducted to detect P4H9 on clinical tissue samples from 156 patients with colorectal cancer. Risk factors for metastases and survival were calculated for clinical implication of PDM-expressing spindle-shaped fibroblasts. Results: Multivariate analysis showed that PDM-expressing spindle-shaped fibroblasts were an independent risk factor for lymph node metastasis, hematogenous metastasis, and poor survival. A Kaplan–Meier survival curve indicated that PDM-expressing spindle-shaped fibroblasts were associated with shorter survival time (P<0.0001). Immunofluorescence showed PDM expression on CCD-18Co fibroblasts and two colorectal cancer cell lines (HCT116 and HCT-15). Conclusions: PDM-expressing spindle-shaped fibroblasts are associated with metastasis and shorter survival in colorectal cancer patients. PDM-expressing spindle-shaped fibroblasts may have a role in eliciting the malignant phenotype of colorectal cancer. PMID:26469833

  19. 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

    2015-01-01

    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.

  20. PPARγ partial agonist GQ-16 strongly represses a subset of genes in 3T3-L1 adipocytes

    SciTech Connect

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

    2015-08-28

    Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor gamma (PPARγ) agonists that improve insulin resistance but trigger side effects such as weight gain, edema, congestive heart failure and bone loss. GQ-16 is a PPARγ 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 PPARγ target genes, or whether it displays gene-selective actions. To determine how GQ-16 influences PPARγ 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. PPARγ 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 PPARγ responsive genes. Strong repressive effects could contribute to physiologic actions of GQ-16. - Highlights: • GQ-16 is an insulin sensitizing PPARγ ligand with reduced harmful side effects. • GQ-16 displays a continuum of weak partial agonist activities at PPARγ-induced genes. • GQ-16 exerts strong repressive effects at a subset of genes. • These inhibitor actions should be evaluated in models of adipose tissue inflammation.

  1. Berberine Alleviates Olanzapine-Induced Adipogenesis via the AMPKα–SREBP Pathway in 3T3-L1 Cells

    PubMed Central

    Li, Yanjie; Zhao, Xiaomin; Feng, Xiyu; Liu, Xuemei; Deng, Chao; Hu, Chang-Hua

    2016-01-01

    The aim of this study was to investigate the mechanisms underlying the inhibitory effects of berberine (BBR) on olanzapine (OLZ)-induced adipogenesis in a well-replicated 3T3-L1 cell model. Oil-Red-O (ORO) staining showed that BBR significantly decreased OLZ-induced adipogenesis. Co-treatment with OLZ and BBR decreased the accumulation of triglyceride (TG) and total cholesterol (TC) by 55.58% ± 3.65% and 49.84% ± 8.31%, respectively, in 3T3-L1 adipocytes accompanied by reduced expression of Sterol regulatory element binding proteins 1 (SREBP1), fatty acid synthase (FAS), peroxisome proliferator activated receptor-γ (PPARγ), SREBP2, low-density lipoprotein receptor (LDLR), and hydroxymethylglutaryl-coenzyme A reductase (HMGR) genes compared with OLZ alone. Consistently, the co-treatment downregulated protein levels of SREBP1, SREBP2, and LDLR by 57.71% ± 9.42%, 73.05% ± 11.82%, and 59.46% ± 9.91%, respectively. In addition, co-treatment reversed the phosphorylation level of AMP-activated protein kinase-α (AMPKα), which was reduced by OLZ, determined via the ratio of pAMPKα:AMPKα (94.1%) compared with OLZ alone. The results showed that BBR may prevent lipid metabolism disorders caused by OLZ by reversing the degree of SREBP pathway upregulated and the phosphorylation of AMPKα downregulated. Collectively, these results indicated that BBR could be used as a potential adjuvant to prevent dyslipidemia and obesity caused by the use of second-generation antipsychotic medication. PMID:27834848

  2. Co-ordinate regulation of the cytoskeleton in 3T3 cells overexpressing thymosin-beta4.

    PubMed

    Golla, R; Philp, N; Safer, D; Chintapalli, J; Hoffman, R; Collins, L; Nachmias, V T

    1997-01-01

    In several cell types, short-term increases in the concentration of the G-actin-sequestering peptide thymosin-beta4 (Tbeta4) cause the disassembly of F-actin bundles. To determine the extent of cell adaptability to these reductions in F-actin, we overexpressed Tbeta4 in NIH 3T3 cells. In cell lines with Tbeta4 levels twice those of vector controls, G-actin increased approximately twofold as expected. However, F-actin did not decrease as in short-term experiments but rather also increased approximately twofold so that the G-F ratio remained constant. Surprisingly, the cytoskeletal proteins myosin IIA, alpha-actinin, and tropomyosin also increased nearly twofold. These increases were specific; DNA, total protein, lactic dehydrogenase, profilin, and actin depolymerizing factor levels were unchanged in the overexpressing cells. The Tbeta4 lines spread more fully and adhered to the dish more strongly than vector controls; this altered phenotype correlated with a twofold increase in talin and alpha5-integrin and a nearly threefold increase in vinculin. Focal adhesions, detected by indirect immunofluorescence with antivinculin, were increased in size over the controls. Northern blotting showed that mRNAs for both beta-actin and vinculin were increased twofold in the overexpressing lines. We conclude that 1) NIH 3T3 cells adapt to increased levels of G-actin sequestered by increased Tbeta4 by increasing their total actin so that the F-actin/G-actin ratio remains constant; 2) these cells coordinately increase several cytoskeletal and adhesion plaque proteins; and 3) at least for actin and vinculin, this regulation is at the transcriptional level. We therefore propose that the proteins of this multimember interacting complex making up the actin-based cytoskeleton, are coordinately regulated by factors that control the expression of several proteins. The mechanism may bear similarities to the control of synthesis of another multimember interacting complex, the myofibril of

  3. Berberine Alleviates Olanzapine-Induced Adipogenesis via the AMPKα-SREBP Pathway in 3T3-L1 Cells.

    PubMed

    Li, Yanjie; Zhao, Xiaomin; Feng, Xiyu; Liu, Xuemei; Deng, Chao; Hu, Chang-Hua

    2016-11-09

    The aim of this study was to investigate the mechanisms underlying the inhibitory effects of berberine (BBR) on olanzapine (OLZ)-induced adipogenesis in a well-replicated 3T3-L1 cell model. Oil-Red-O (ORO) staining showed that BBR significantly decreased OLZ-induced adipogenesis. Co-treatment with OLZ and BBR decreased the accumulation of triglyceride (TG) and total cholesterol (TC) by 55.58% ± 3.65% and 49.84% ± 8.31%, respectively, in 3T3-L1 adipocytes accompanied by reduced expression of Sterol regulatory element binding proteins 1 (SREBP1), fatty acid synthase (FAS), peroxisome proliferator activated receptor-γ (PPARγ), SREBP2, low-density lipoprotein receptor (LDLR), and hydroxymethylglutaryl-coenzyme A reductase (HMGR) genes compared with OLZ alone. Consistently, the co-treatment downregulated protein levels of SREBP1, SREBP2, and LDLR by 57.71% ± 9.42%, 73.05% ± 11.82%, and 59.46% ± 9.91%, respectively. In addition, co-treatment reversed the phosphorylation level of AMP-activated protein kinase-α (AMPKα), which was reduced by OLZ, determined via the ratio of pAMPKα:AMPKα (94.1%) compared with OLZ alone. The results showed that BBR may prevent lipid metabolism disorders caused by OLZ by reversing the degree of SREBP pathway upregulated and the phosphorylation of AMPKα downregulated. Collectively, these results indicated that BBR could be used as a potential adjuvant to prevent dyslipidemia and obesity caused by the use of second-generation antipsychotic medication.

  4. Cellular uptake and fate of fibroin microspheres loaded with randomly fragmented DNA in 3T3 cells.

    PubMed

    Lee, Jin Sil; Hur, Won

    2016-01-01

    Purified fibroin protein can be obtained in large quantities from silk fibers and processed to form microscopic particles as delivery vehicles for therapeutic agents. In this study, we demonstrated that fibroin microspheres were taken up by 3T3 cells, localized in the nonlysosomal compartment, and secreted from the cytoplasm after medium replenishment. DNA-loaded microspheres were taken up by >95% of 3T3 cells. DNA cargo had no influence on the intracellular trafficking of microspheres, while fluorescently labeled cargo DNA was observed in the lysosomal compartment and in the microspheres. These results indicate that fibroin microspheres can travel through 3T3 cells without making any contact with the lysosomal compartments. The amount of DNA loaded in the microspheres taken up by 3T3 cells was estimated up to 831.0 pg/cell. Thus, fibroin microspheres can deliver a large amount of randomly fragmented DNA (<10 kb) into the cytoplasmic compartment of 3T3 cells.

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

    PubMed

    Zhang, Yi; Qian, Qian; Ge, Dandan; Li, Yuhong; Wang, Xinrui; Chen, Qiu; Gao, Xiumei; Wang, Tao

    2011-11-09

    A 70% ethanol-water extract from the leaves of Mangifera indica L. (Anacardiaceae) inhibited triglyceride (TG) accumulation in 3T3-L1 cells. From the active fraction, seven new benzophenone C-glycosides, foliamangiferosides A (1), A(1) (2), A(2) (3), B (4), C(1) (5), C(2) (6), and C(3) (7), together with five known compounds were isolated and the structures were elucidated on the basis of chemical and physicochemical evidence. The effects of these compounds on TG and the free fatty acid level in 3T3-L1 cells were determined, and the structure-activity relationship was discussed. On the basis of the AMPK signaling pathway, several compounds were found to increase the AMPK enzyme expression and down-regulate lipogenic enzyme gene expression such as SREBP1c, FAS, and HSL.

  6. Emdogain-regulated gene expression in palatal fibroblasts requires TGF-βRI kinase signaling.

    PubMed

    Stähli, Alexandra; Bosshardt, Dieter; Sculean, Anton; Gruber, Reinhard

    2014-01-01

    Genome-wide microarrays have suggested that Emdogain regulates TGF-β target genes in gingival and palatal fibroblasts. However, definitive support for this contention and the extent to which TGF-β signaling contributes to the effects of Emdogain has remained elusive. We therefore studied the role of the TGF-β receptor I (TGF-βRI) kinase to mediate the effect of Emdogain on palatal fibroblasts. Palatal fibroblasts were exposed to Emdogain with and without the inhibitor for TGF-βRI kinase, SB431542. Emdogain caused 39 coding genes to be differentially expressed in palatal fibroblasts by microarray analysis (p<0.05; >10-fold). Importantly, in the presence of the TGF-βRI kinase inhibitor SB431542, Emdogain failed to cause any significant changes in gene expression. Consistent with this mechanism, three independent TGF-βRI kinase inhibitors and a TGF-β neutralizing antibody abrogated the increased expression of IL-11, a selected Emdogain target gene. The MAPK inhibitors SB203580 and U0126 lowered the impact of Emdogain on IL-11 expression. The data support that TGF-βRI kinase activity is necessary to mediate the effects of Emdogain on gene expression in vitro.

  7. 3T3 cell motility and morphology before, during, and after exposure to extremely-low-frequency magnetic fields

    SciTech Connect

    Spadinger, I.; Palcic, B.; Agnew, D.

    1995-08-01

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

  8. 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

  9. p53/PUMA expression in human pulmonary fibroblasts mediates cell activation and migration in silicosis

    PubMed Central

    Wang, Wei; Liu, Haijun; Dai, Xiaoniu; Fang, Shencun; Wang, Xingang; Zhang, Yingming; Yao, Honghong; Zhang, Xilong; Chao, Jie

    2015-01-01

    Phagocytosis of SiO2 into the lung causes an inflammatory cascade that results in fibroblast proliferation and migration, followed by fibrosis. Clinical evidence has indicated that the activation of alveolar macrophages by SiO2 produces rapid and sustained inflammation characterized by the generation of monocyte chemotactic protein 1, which, in turn, induces fibrosis. However, the details of events downstream of monocyte chemotactic protein 1 activity in pulmonary fibroblasts remain unclear. Here, to elucidate the role of p53 in fibrosis induced by silica, both the upstream molecular mechanisms and the functional effects on cell proliferation and migration were investigated. Experiments using primary cultured adult human pulmonary fibroblasts led to the following results: 1) SiO2 treatment resulted in a rapid and sustained increase in p53 and PUMA protein levels; 2) the MAPK and PI3K pathways were involved in the SiO2-induced alteration of p53 and PUMA expression; and 3) RNA interference targeting p53 and PUMA prevented the SiO2-induced increases in fibroblast activation and migration. Our study elucidated a link between SiO2-induced p53/PUMA expression in fibroblasts and cell migration, thereby providing novel insight into the potential use of p53/PUMA in the development of novel therapeutic strategies for silicosis treatment. PMID:26576741

  10. 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

  11. Downregulated miR-29a/b/c during Contact Inhibition Stage Promote 3T3-L1 Adipogenesis by Targeting DNMT3A

    PubMed Central

    Jia, Yudong; Zhang, Ying; Tang, Yanfeng; Li, Wenlong; Fan, Yanan; Zhang, Xiaodong; Liu, Youwen

    2017-01-01

    Differentiation of 3T3-L1 cells into adipocytes involves a highly-orchestrated series of events including contact inhibition (CI), clonal expansion, growth arrest, and terminal differentiation. Recent study demonstrated that 3T3-L1 preadipocytes will not be differentiated into mature adipocytes without CI stage, which indicated that CI stage plays an important role during 3T3-L1 adipogenesis. However, the molecular mechanism is not yet fully understood. In the present study, we found that the expression level of miR-29a/b/c was decreased and the expression of DNMT3A was up-regulated during CI stage, respectively. Furthermore, overexpression of miR-29a/b/c during CI stage inhibits adipogenesis significantly but not at other stages. In addition, miR-29a/b/c repressed DNMT3A expression by directly targeting its 3’ untranslated region (3’ UTR). Our data reveal a novel mechanism of miR-29a/b/c in the regulation of adipogenesis. PMID:28114345

  12. Influence of Mechanical Force on Bone Matrix Proteins in Ovariectomised Mice and Osteoblast-like MC3T3-E1 Cells

    PubMed Central

    ZHANG, MENG; ISHIKAWA, SHINTARO; INAGAWA, TOMOKO; IKEMOTO, HIDESHI; GUO, SHIYU; SUNAGAWA, MASATAKA; HISAMITSU, TADASHI

    2017-01-01

    Aim: To investigate the effect of mechanical stress on periostin and semaphorin-3A expression in a murine model of postmenopausal osteoporosis and in osteoblast-like MC3T3-E1 cells. Materials and Methods: Female mice were divided into three groups and treated with a sham operation, ovariectomy (OVX) or OVX plus treadmill training (OVX+Run). After 10 weeks, tibias were used for histological analysis. MC3T3-E1 cells were burdened by mechanical stress using a centrifuge or were treated with periostin, and the production of biologically-active semaphorin-3A was examined in vitro. Results: In OVX+Run group tibias, the number of tartrate-resistant acid phosphatase-positive osteoclasts was lower than in the OVX group, and the expression of periostin and semaphorin-3A was higher. In MC3T3-E1 cells, centrifugal stress significantly increased periostin and semaphorin-3A mRNA expression. Treatment with periostin increased the semaphorin-3A level. Conclusion: We speculate that mechanical load may increase periostin production in osteoblasts, and periostin may inhibit osteoclast differentiation by its effects on semaphorin-3A. Our results support the concept of a positive correlation between exercise and inhibition of osteoclasts in post-menopausal osteoporosis. PMID:28064225

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

    PubMed

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

    2012-05-01

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

  14. Dihydrocytochalasin B. Biological effects and binding to 3T3 cells

    PubMed Central

    Atlas, S. J.; Lin, S.

    1978-01-01

    Dihydrocytochalasin B (H2CB) does not inhibit sugar uptake in BALB/c 3T3 cells. Excess H2CB does not affect inhibition of sugar uptake by cytochalasin B (CB), indicating that it does not compete with CB for binding to high-affinity sites. As in the case of CB, H2CB inhibits cytokinesis and changes the morphology of the cells. These results demonstrate that the effects of CB on sugar transport and on cell motility and morphology involve separate and independent sites. Comparison of the effects of H2CB, CB, and cytochalasin D (CD) indicates that treatment of cells with any one of the compounds results in the same series of morphological changes; the cells undergo zeiosis and elongation at 2-4 microM CB and become arborized and rounded up at 10-50 microM CB. H2CB is slightly less potent than CB, whereas CD is five to eight times more potent than CB in causing a given state of morphological change. These results indicate that the cytochalasin-induced changes in cell morphology are mediated by a specific site(s) which can distinguish the subtle differences in the structures of the three compounds. Competitive binding studies indicate that excess H2CB displaces essentially all of the high-affinity bound [3H]CB, but, at less than 5 x 10(-5) M H2CB is not so efficient as unlabeled CB in the displacement reaction. In contrast, excess CD displaces up to 40% of the bound [3H]CB. These results suggest that three different classes of high-affinity CB binding sites exist in 3T3 cells: sites related to sugar transport, sites related to cell motility and morphology, and sites with undetermined function. PMID:10605443

  15. Genetic polymorphism directs IL-6 expression in fibroblasts but not selected other cell types.

    PubMed

    Noss, Erika H; Nguyen, Hung N; Chang, Sook Kyung; Watts, Gerald F M; Brenner, Michael B

    2015-12-01

    Interleukin (IL)-6 blockade is an effective treatment for rheumatoid arthritis (RA), and synovial fibroblasts are a major IL-6 producer in the inflamed joint. We found that human RA and osteoarthritis (OA) synovial fibroblasts derived from independent donors reproducibly segregated into low, medium, and high IL-6 producers, independent of stimulus, cell passage, or disease state. IL-6 expression pattern correlated strongly with total mRNA expression, not mRNA stability, suggesting transcriptional rather than posttranscriptional regulation. High-fibroblast IL-6 expression was significantly associated with the IL-6 proximal promoter single nucleotide polymorphism (SNP) rs1800795 minor allele (CC) genotype. In contrast, no association between this SNP and IL-6 production was detected in CD14(+) monocytes, another major producer of synovial IL-6. Luciferase expression assays confirmed that this SNP was associated with differential IL-6 expression in fibroblasts. To date, several association studies examining rs1800795 allele frequency and disease risk have reported seemingly conflicting results ranging from no association to association with either the major or minor allele across a spectrum of conditions, including cancer and autoimmune, cardiovascular, infectious, and metabolic diseases. This study points to a prominent contribution from promoter genetic variation in fibroblast IL-6 regulation, but not in other IL-6-producing cell types. We propose that some of the heterogeneity in these clinical studies likely reflects the cellular source of IL-6 in specific diseases, much of which may be produced by nonhematopoietic cells. These results highlight that functional analysis of disease-associated SNPs on gene expression and pathologic processes must consider variation in diverse cell types.

  16. Problem-Based Test: The Effect of Fibroblast Growth Factor on Gene Expression

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2011-01-01

    This paper shows the results of an experiment in which the effects of fibroblast growth factor (FGF), actinomycin D (Act D; an inhibitor of transcription), and cycloheximide (CHX; an inhibitor of translation) were studied on the expression of two genes: a gene called "Fnk" and the gene coding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).…

  17. Bcl-2 expression in synovial fibroblasts is essential for maintaining mitochondrial homeostasis and cell viability.

    PubMed

    Perlman, H; Georganas, C; Pagliari, L J; Koch, A E; Haines, K; Pope, R M

    2000-05-15

    The regulation of proliferation and cell death is vital for homeostasis, but the mechanism that coordinately balances these events in rheumatoid arthritis (RA) remains largely unknown. In RA, the synovial lining thickens in part through increased proliferation and/or decreased synovial fibroblast cell death. Here we demonstrate that the anti-apoptotic protein, Bcl-2, is highly expressed in RA compared with osteoarthritis synovial tissues, particularly in the CD68-negative, fibroblast-like synoviocyte population. To determine the importance of endogenous Bcl-2, an adenoviral vector expressing a hammerhead ribozyme to Bcl-2 (Ad-Rbz-Bcl-2) mRNA was employed. Ad-Rbz-Bcl-2 infection resulted in reduced Bcl-2 expression and cell viability in synovial fibroblasts isolated from RA and osteoarthritis synovial tissues. In addition, Ad-Rbz-Bcl-2-induced mitochondrial permeability transition, cytochrome c release, activation of caspases 9 and 3, and DNA fragmentation. The general caspase inhibitor zVAD.fmk blocked caspase activation, poly(ADP-ribose) polymerase cleavage, and DNA fragmentation, but not loss of transmembrane potential or viability, indicating that cell death was independent of caspase activation. Ectopically expressed Bcl-xL inhibited Ad-Rbz-Bcl-2-induced mitochondrial permeability transition and apoptosis in Ad-Rbz-Bcl-2-transduced cells. Thus, forced down-regulation of Bcl-2 does not induce a compensatory mechanism to prevent loss of mitochondrial integrity and cell death in human fibroblasts.

  18. Molecular cloning and characterization of plastin, a human leukocyte protein expressed in transformed human fibroblasts.

    PubMed Central

    Lin, C S; Aebersold, R H; Kent, S B; Varma, M; Leavitt, J

    1988-01-01

    The phosphoprotein plastin was originally identified as an abundant transformation-induced polypeptide of chemically transformed neoplastic human fibroblasts. This abundant protein is normally expressed only in leukocytes, suggesting that it may play a role in hemopoietic cell differentiation. Protein microsequencing of plastin purified from leukemic T lymphocytes by high-resolution two-dimensional gel electrophoresis produced eight internal oligopeptide sequences. An oligodeoxynucleotide probe corresponding to one of the oligopeptides was used to clone cDNAs from transformed human fibroblasts that encoded the seven other oligopeptides predicted for human plastin. Sequencing and characterization of two cloned cDNAs revealed the existence of two distinct, but closely related, isoforms of plastin--l-plastin, which is expressed in leukocytes and transformed fibroblasts, and t-plastin, which is expressed in normal cells of solid tissues and transformed fibroblasts. The leukocyte isoform l-plastin is expressed in a diverse variety of human tumor cell lines, suggesting that it may be involved in the neoplastic process of some solid human tumors. Images PMID:3211125

  19. In vitro MC3T3 osteoblast adhesion with respect to surface roughness of Ti6Al4V substrates.

    PubMed

    Linez-Bataillon, P; Monchau, F; Bigerelle, M; Hildebrand, H F

    2002-08-01

    This work investigates the role of the surface roughness of Ti6Al4V on the cell morphology, proliferation and adhesion, and in particular on the variation of the expression of cell adhesion proteins. Standardised test samples with five different surface preparations are used: sandblasted, 80, 1200, and 4000 grade polished, mirror polished. Surface roughness is analysed by Scanning Electron Microscopy and LASER Confocal Microscopy. Cell culture experiments are performed with MC3T3-E1 mouse osteoblasts after 3 days culture: proliferation rate, morphology and adhesion are assessed. The variations of expression of cell adhesion proteins are evidenced by indirect immune fluorescence method: actin from the cytoskeleton, vinculin from the focal adhesion complex, fibronectin and collagen I from the extracellular matrix. The results reveal a clear influence of surface roughness of Ti6Al4V on cell proliferation, morphology and adhesion. A significant correlation is established between surface roughness and cell growth. More the surface is smooth more the osteoblasts proliferate and appear spread out on the test samples. In addition, the expression of adhesion proteins varies with respect to the surface roughness. These results indicate a direct relationship between the decrease of cell adhesion and the increase of cell proliferation on mirror polished materials.

  20. Adipogenesis, lipogenesis and lipolysis is stimulated by mild but not severe hypoxia in 3T3-L1 cells.

    PubMed

    Weiszenstein, Martin; Musutova, Martina; Plihalova, Andrea; Westlake, Katerina; Elkalaf, Moustafa; Koc, Michal; Prochazka, Antonin; Pala, Jan; Gulati, Sumeet; Trnka, Jan; Polak, Jan

    2016-09-16

    In-vitro investigation of the effects of hypoxia is limited by physical laws of gas diffusion and cellular O2 consumption, making prolonged exposures to stable O2 concentrations impossible. Using a gas-permeable cultureware, chronic effects of mild and severe hypoxia on triglyceride accumulation, lipid droplet size distribution, spontaneous lipolysis and gene expression of adipocyte-specific markers were assessed. 3T3-L1 cells were differentiated under 20%, 4% or 1% O2 using a gas-permeable cultureware. Triglyceride accumulation, expression of genes characteristic for advanced adipocyte differentiation and involvement of key lipogenesis enzymes were assessed after exposures. Lipogenesis increased by 375% under mild hypoxia, but dropped by 43% in severe hypoxia. Mild, but not severe, hypoxia increased formation of large lipid droplets 6.4 fold and strongly induced gene expression of adipocyte-specific markers. Spontaneous lipolysis increased by 488% in mild, but only by 135% in severe hypoxia. Inhibition of ATP-dependent citrate lyase suppressed hypoxia-induced lipogenesis by 81% and 85%. Activation of HIF inhibited lipogenesis by 59%. Mild, but not severe, hypoxia stimulates lipolysis and promotes adipocyte differentiation, probably through excess of acetyl-CoA originating from tricarboxylic acid cycle independently of HIF activation.

  1. Effects of high glucose on caveolin-1 and insulin signaling in 3T3-L1 adipocytes

    PubMed Central

    Palacios-Ortega, Sara; Varela-Guruceaga, Maider; Martínez, J. Alfredo; de Miguel, Carlos; Milagro, Fermín I.

    2016-01-01

    ABSTRACT Adipocytes exposed to high glucose concentrations exhibit impaired metabolic function, including an increase of oxidative and proinflammatory factors that might favor the development of insulin resistance. Caveolin-1 (Cav-1) is a key mediator of the insulin transduction pathway whose expression is significantly enhanced during adipocyte differentiation. In this work, we studied the effects of high glucose concentration on the regulation of Cav-1 expression and activation and its relation to the insulin signaling pathway during the adipogenic process and in long-term differentiated adipocytes. Both, long-term high glucose exposure during adipogenesis and short-term glucose incubation of mature adipocytes, promoted triglyceride accumulation in 3T3-L1 cells. The short-term exposure of mature adipocytes to high glucose significantly reduced the sensitivity to insulin of Cav-1, insulin receptor (IR) and potein kinase B (AKT-2) phosphorylation, as well as insulin-induced deoxyglucose uptake. Adipocytes differentiated in the presence of high glucose lost Cav-1 and IR response to insulin-stimulated phosphorylation, but maintained the insulin sensitivity of AKT-2 phosphorylation and deoxyglucose uptake. Although long-term high glucose exposure increased DNA methylation in Cav-1 promoter, Cav-1 expression was not affected. Moreover, these cells showed an increase of Cav-1, IR and AKT-2 protein content, pointing to an adaptive response induced by the long-term high glucose exposure. PMID:27144098

  2. Effects of high glucose on caveolin-1 and insulin signaling in 3T3-L1 adipocytes.

    PubMed

    Palacios-Ortega, Sara; Varela-Guruceaga, Maider; Martínez, J Alfredo; de Miguel, Carlos; Milagro, Fermín I

    2016-01-01

    Adipocytes exposed to high glucose concentrations exhibit impaired metabolic function, including an increase of oxidative and proinflammatory factors that might favor the development of insulin resistance. Caveolin-1 (Cav-1) is a key mediator of the insulin transduction pathway whose expression is significantly enhanced during adipocyte differentiation. In this work, we studied the effects of high glucose concentration on the regulation of Cav-1 expression and activation and its relation to the insulin signaling pathway during the adipogenic process and in long-term differentiated adipocytes. Both, long-term high glucose exposure during adipogenesis and short-term glucose incubation of mature adipocytes, promoted triglyceride accumulation in 3T3-L1 cells. The short-term exposure of mature adipocytes to high glucose significantly reduced the sensitivity to insulin of Cav-1, insulin receptor (IR) and potein kinase B (AKT-2) phosphorylation, as well as insulin-induced deoxyglucose uptake. Adipocytes differentiated in the presence of high glucose lost Cav-1 and IR response to insulin-stimulated phosphorylation, but maintained the insulin sensitivity of AKT-2 phosphorylation and deoxyglucose uptake. Although long-term high glucose exposure increased DNA methylation in Cav-1 promoter, Cav-1 expression was not affected. Moreover, these cells showed an increase of Cav-1, IR and AKT-2 protein content, pointing to an adaptive response induced by the long-term high glucose exposure.

  3. Inhibitory effects of compounds isolated from the dried branches and leaves of murta (Myrceugenia euosma) on lipid accumulation in 3T3-L1 cells.

    PubMed

    Oikawa, Naoki; Nobushi, Yasuhito; Wada, Taira; Sonoda, Kumiko; Okazaki, Yuzo; Tsutsumi, Shigetoshi; Park, Yong Kun; Kurokawa, Masahiko; Shimba, Shigeki; Yasukawa, Ken

    2016-07-01

    As obesity is a global health concern the demand for anti-obesity drugs is high. In this study, we investigated the anti-obesity effect of the dried branches and leaves of murta (Myrceugenia euosma Legrand, Myrtaceae). A methanol extract of the dried branches and leaves of murta inhibited adipogenesis in 3T3-L1 cells. Three known flavanones-cryptostrobin (1), pinocembrin (4), and 5,7-dihydroxy-6,8-dimethylflavanone (6), and three chalcones-2',6'-dihydroxy-3'-methyl-4'-methoxychalcone (2), pinostrobin chalcone (3), and 2',6'-dihydroxy-4'-methoxy-3',5'-dimethylchalcone (5) were isolated from the active fraction. Structures of these compounds were identified using various spectral data. Each of these compounds also inhibited adipogenesis in 3T3-L1 cells. In particular, compound 3 was a more potent inhibitor of triglyceride accumulation than the positive control berberine. Gene expression studies revealed that treatment of 3T3-L1 cells with 3 lowers the expression levels of CCAAT/enhancer-binding protein α and peroxisome proliferator activator γ2 during adipogenesis without affecting cell viability. Treatment of 3T3-L1 cells with 3 reduced the expression levels of mRNAs encoding sterol regulatory element-binding protein 1c and several lipogenic enzymes, including fatty acid synthase and stearoyl CoA desaturase-1. These results indicate that the methanol extract and compounds isolated from the dried branches and leaves of murta exert their anti-obesity effects through the inhibition of adipogenesis.

  4. Bradykinin promotes Toll like receptor-4 expression in human gingival fibroblasts.

    PubMed

    Gutiérrez-Venegas, Gloria; Arreguín-Cano, Juan Antonio; Hernández-Bermúdez, Cristina

    2012-12-01

    Bacterial infections are a potent mechanism for enzymatic generation of kinins such as bradykinin (BK), a universal mediator for inducing inflammatory reaction by associating with the B2 receptor and stimulating liberation of arachidonic acid and synthesis of prostaglandin E2 (PGE2). In this study we evaluate the role of bradykinin in regulating the expression of TLR4 receptor in human gingival fibroblasts. We examine the ability of bradykinin to modulate inflammatory response of human gingival fibroblasts to Gram-negative components and evaluated the role of Toll-like receptors (TLR)-4 in the co-operation between bradykinin and bacterial pathogens. We show that treatment with bradykinin promotes TLR4 receptor expression in human gingival fibroblasts (HGF) and amplifies inflammatory responses to the bacterial components of Gram-negative bacteria. The TLR4 expression induced by bradykinin was blocked with Hoe 140, a B2R antagonist. When HGF cells were incubated with BK resulted of an increased in cyclooxygenase-2 (COX-2) expression and prostaglandin E2 synthesis. Bradykinin and lipopolysaccharide, a specific TLR4 ligand stimulated COX-2 expression. In other series of experiments we found that ERK, phosphatidylinositol-3 kinase, protein kinase C and NFkB are involved in BK promoted-increased in TLR4 expression. The results demonstrate that bradykinin up-regulates the expression of TLR4 and promotes an additive increase in inflammatory responses to lipopolysaccharides.

  5. Relaxin Modulates the Expression of MMPs and TIMPs in Fibroblasts of Patients with Carpal Tunnel Syndrome

    PubMed Central

    Kang, Young-Mi; Lee, Hwan-Mo; Moon, Seong-Hwan; Kang, Ho

    2017-01-01

    Purpose The aim of this study was to investigate the anti-fibrotic effect of relaxin in subsynovial fibroblasts activated by transforming growth factor beta (TGF-β). Materials and Methods To test the anti-fibrotic effect of an adenovirus-relaxin construct (Ad-RLN) on subsynovial fibroblasts in vitro, cells from subsynovial connective tissue of patients with carpal tunnel syndrome were activated with TGF-β1 and exposed to Ad-RLN (as a therapeutic gene) or adenovirus-lacZ construct (as a marker gene) for four hours. Subsynovial fibroblast cultures without adenoviral exposure served as controls. Results We observed induction of gene expressions of collagen I, III and IV, as well as the abatement of alpha-smooth muscle actin (a-SMA) synthesis, Smad2 phosphorylation, and fibronectin at the protein level, in comparison to controls. In addition, protein expressions of matrix metalloproteinase (MMP) I was significantly induced, whereas the protein expressions of tissue inhibitor of metalloproteinases (TIMP) I and IV were reduced due to relaxin expression. Conclusion RLN prevents excessive synthesis of extracellular matrix by reducing the expressions of its components, such as fibronectin, a-SMA, and phosphorylated Smad2, by increasing the expression of MMPs; and by decreasing the expression of TIMPs. PMID:28120574

  6. ALK5 inhibition blocks TGFβ-induced CCN1 expression in human foreskin fibroblasts.

    PubMed

    Thompson, Katherine; Murphy-Marshman, Hannah; Leask, Andrew

    2014-03-01

    The potent profibrotic cytokine TGFβ induces connective tissue growth factor (CCN2/CTGF) is induced in fibroblasts in a fashion sensitive to SB-431542, a specific pharmacological inhibitor of TGFβ type I receptor (ALK5). In several cell types, TGFβ induces CCN1 but suppresses CCN3, which opposes CCN1/CCN2 activities. However, whether SB-431542 alters TGFβ-induced CCN1 or CCN3 in human foreskin fibroblasts in unclear. Here we show that TGFβ induces CCN1 but suppresses CCN3 expression in human foreskin fibroblasts in a SB-431542-sensitive fashion. These results emphasize that CCN1/CCN2 and CCN3 are reciprocally regulated and support the notion that blocking ALK5 or addition of CCN3 may be useful anti-fibrotic approaches.

  7. Basic calcium phosphate crystal-induced Egr-1 expression stimulates mitogenesis in human fibroblasts

    SciTech Connect

    Zeng, Xiao R.; Sun Yubo; Wenger, Leonor; Cheung, Herman S. . E-mail: hcheung@med.miami.edu

    2005-05-13

    Previously, we have reported that basic calcium phosphate (BCP) crystals stimulate mitogenesis and synthesis of matrix metalloproteinases in cultured human foreskin and synovial fibroblasts. However, the detailed mechanisms involved are still unclear. In the present study, using RT-PCR and Egr-1 promoter analysis we showed that BCP crystals could stimulate early growth response gene Egr-1 transcription through a PKC{alpha}-dependent p44/p42 MAPK pathway. Using a retrovirus gene expression system (Clontech) to overexpress Egr-1 in human fibroblast BJ-1 cells resulted in promotion of mitogenesis measured either by MTT cell proliferation analysis or by direct cell counting. The results demonstrate that Egr-1 may play a key role in mediating BCP crystal-induced synovial fibroblast mitogenesis.

  8. Effect of Fermented Red Ginseng Extract Enriched in Ginsenoside Rg3 on the Differentiation and Mineralization of Preosteoblastic MC3T3-E1 Cells

    PubMed Central

    Siddiqi, Muhammad Zubair; Siddiqi, Muhammad Hanif; Jin, Yan; Huq, Md. Amdadul

    2015-01-01

    Abstract In this study, red ginseng extract (RGE) was converted into high-content minor ginsenosides by fermenting with Bgp1 enzymes at 37°C for 5 days. Compared to the RGE, the minor ginsenoside contents were increased in fermented red ginseng extract (FRGE). Moreover, the amount of minor ginsenosides such as Rh1 (11%) and Rg2 (16%) was slightly augmented, while the level of Rg3 (33%) was significantly increased after bioconversion. Furthermore, we also examined and compared the effect of RGE and FRGE on the differentiation and mineralization of preosteoblastic MC3T3-E1 cells. Similarly, the level of mRNA expression of intracellular alkaline phosphatase (ALP) activity, type-1 collagen (Col-I) was also increased. Based on the comparison, it is clear that the FRGE has improved effects on bone formation and differentiation of preosteoblastic MC3T3-E1 cells. PMID:25764149

  9. 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

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

    SciTech Connect

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

    1987-06-01

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

  11. Enhanced osteogenic differentiation of MC3T3-E1 cells on grid-topographic surface and evidence for involvement of YAP mediator.

    PubMed

    Zhang, Yingying; Gong, He; Sun, Yan; Huang, Yan; Fan, Yubo

    2016-05-01

    Numerous studies have shown that surface topography can promote cell-substrate associations and deeply influence cell fate. The intracellular mechanism or how micro- or nano-patterned extracellular signal is ultimately linked to activity of nuclear transcription factors remains unknown. It has been reported that Yes-associated protein (YAP) can respond to extracellular matrix microenvironment signals, thus regulates stem cell differentiation process. We propose that YAP may play a role in mediating the topography induced cell differentiation. To this end, we fabricated polydimethylsiloxane (PDMS) micropatterns with grid topology (GT) (3 μm pattern width, 2 μm pattern interval length, 7 μm pattern height); nonpatterned PDMS substrates were used as the planar controls. The MC3T3-E1 cells were then cultured on these surfaces, respectively, in osteogenic inducing medium. Cell differentiation in terms of osteogenesis related gene expression, protein levels, alkaline phosphatase activity and extracellular matrix mineralization was assessed. It was shown that the cells on GT surfaces had stronger osteogenesis capacity. In addition, expression level of YAP was increased when MC3T3-E1 cells grew on GT substrates, which was similar to the levels of osteogenic differentiation markers. It was also shown that YAP knockdown attenuated GT substrates-induced MC3T3-E1 differentiation, which reduced the osteogenic differentiation effect of the GT substrates. Collectively, our findings indicate that GT substrates-induced MC3T3-E1 differentiation may be associated with YAP. This paper provides new target points for transcriptional mechanism research of microenvironment induced cell differentiation and a useful approach to obtain more biofunctionalization scaffolds for tissue engineering.

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

    PubMed

    Lee, Youngyi; Bae, Eun Ju

    2013-11-01

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

  13. Synergistic induction of insulin resistance by endothelin-1 and cAMP in 3T3-L1 adipocytes.

    PubMed

    Chai, Shin-Pei; Fong, Jim C

    2015-10-01

    Both endothelin-1 (ET-1) and cAMP are implicated for inducing insulin resistance. Since we have shown previously that there is a crosstalk between ET-1 and cAMP signaling pathways in regulating glucose uptake in 3T3-L1 adipocytes, we extended our investigation in this study on whether they may have a synergistic effect on inducing insulin resistance. Our results showed that it was indeed the case. Insulin-stimulated glucose uptake, phosphorylation of PKB, IRS-1-associated PI3K, and IRS-1 tyrosine phosphorylation were all inhibited by ET-1 and 8-bromo cAMP in a synergistic manner. IRS-1 protein levels were similarly decreased by ET-1 and 8-bromo cAMP, attributable to suppressed mRNA expression. In addition, after correction for the loss in IRS-1 protein, the inhibition of insulin-stimulated IRS-1 tyrosine phosphorylation or IRS-1-associated PI3K was mainly caused by cAMP. Moreover, whereas IRS-2 protein levels were increased by cAMP or ET-1 plus cAMP, insulin-stimulated IRS-2-associated PI3K activities were abolished by both treatments. Furthermore, ET-1 and β-adrenergic agonists had similar synergistic inhibition on insulin-stimulated glucose uptake. In conclusion, we have shown that ET-1 and cAMP may synergistically induce insulin resistance in adipocytes via inhibiting IRS-1 expression as well as insulin-stimulated IRS-1/IRS-2 activities.

  14. The effect of cultureware surfaces on functional and structural components of differentiated 3T3-L1 preadipocytes.

    PubMed

    Pavlikova, Nela; Weiszenstein, Martin; Pala, Jan; Halada, Petr; Seda, Ondrej; Elkalaf, Moustafa; Trnka, Jan; Kovar, Jan; Polak, Jan

    2015-12-01

    Experiments using cultured primary cells or cell lines are a routine in vitro approach used across multiple biological disciplines, However, the structural and functional influences of various cultureware materials on cultured cells is not clearly understood. Surface treatments of cultureware have proven to have profound effects on cell viability and proliferation. In this study, we investigated the impact of polystyrene and fluorocarbon cultureware dishes on the proteomic profile of differentiated 3T3-L1 preadipocytes. After expansion and differentiation of cells on appropriate cultureware dishes, cell lysates were separated using two-dimensional gel electrophoresis and proteins were visualized with Coomassie blue staining. Spots with the highest differential expression between the two culture conditions were subsequently analyzed using matrix-assisted laser desorption/ionization mass spectrometry and the identified proteins were subjected to pathway analysis. We observed that 43% of all spots were differentially expressed depending on the cultureware. Pathway analysis revealed that glucose metabolism, mitochondrial structure and cell differentiation, represented by 14-3-3 protein-mediated signaling and the mitochondrial inner membrane organizing system (MINOS), were significantly affected by cultureware material. These results indicate that cultureware material can have a profound effect on key adipocyte functional pathways. These effects modifications of the cells should be reflected in the design of in vitro experiments and interpretation of their results.

  15. Transcriptional and translational control of cytoplasmic proteins after serum stimulation of quiescent Swiss 3T3 cells.

    PubMed Central

    Thomas, G; Thomas, G; Luther, H

    1981-01-01

    The synthesis of cytoplasmic proteins from quiescent and serum-stimulated Swiss 3T3 cells was compared by two-dimensional polyacrylamide gel electrophoresis. Four new proteins of Mrs 26,000, 28,000, 45,000, and 47,000 designated N26, N28, N45, and N47, which were not detectable in quiescent cells, appeared 60 min after addition of serum. During the same period, the amount of [35S]methionine incorporated into 10 proteins present in quiescent cells, ranging in Mr from 23,000 to 98,000 and designated Q23-98, increased up to 6-fold, whereas the amount incorporated into three other proteins decreased by a factor of approximately 2. Of the new proteins, N26 was no longer detectable, and the amount of [35S]methionine incorporated into N47 was significantly reduced by 150 min. During this same time, a fifth new protein, N56, appeared, and there was a large increase in the amount of radioactivity incorporated into another protein, Q121. The increases in nine of the proteins were either strongly or completely inhibited by actinomycin D, arguing that the expression of these proteins was under transcriptional control. In contrast, the increases in seven other proteins were unaffected by actinomycin D, suggesting that their expression was under translational control. These proteins will serve as useful markers for determining how cells progress through early lag phase. Images PMID:6946510

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

    PubMed

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

    2012-01-01

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

  17. 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 γ (PPARγ), CCAAT/enhancer binding proteins α (C/EBPα), and δ (C/EBPδ) 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

  18. Expression of integrins by human periodontal ligament and gingival fibroblasts and their involvement in fibroblast adhesion to enamel matrix-derived proteins.

    PubMed

    van der Pauw, M T M; Everts, V; Beertsen, W

    2002-10-01

    We showed recently that human periodontal ligament (PDL) and gingival fibroblasts adhere and spread on enamel matrix protein (EMP) coatings. In the present study, we investigated whether this interaction can be attributed to integrin expression. Human PDL and gingival fibroblasts were cultured for periods up to 24 h on EMP coatings, in the presence of synthetic RGD-containing peptide or an antibody against the beta1 integrin subunit. The cells were first cultured for 24 h under serum-free conditions and then cultured on EMP coatings for 48 h. Integrin expression levels were assessed by flow cytometry analysis. It was found that attachment and spreading on EMP was inhibited by the synthetic RGD-containing peptide, but not by a synthetic RGE-peptide. Both PDL and gingival fibroblasts showed expression of the integrin subunits, alpha2, alpha5, beta1, and the integrin, alphavbeta3. Incubation with an antibody against the beta1 subunit significantly inhibited the attachment and spreading of PDL and gingival fibroblasts on EMP coatings. We conclude that integrins are involved in the interaction of PDL and gingival fibroblasts with EMP.

  19. Effect of diacylglycerol acyltransferase 2 overexpression in 3T3-L1 is associated to an increase in mono-unsaturated fatty acid accumulation

    PubMed Central

    2014-01-01

    Background Fatty acid (FA) composition is the most important parameter affecting the flavor and nutritional value of the meat. The final and the only committed step in the biosynthesis of triglycerides is catalyzed by diacylglycerol acyltransferase 2 (DGAT2). The role of DGAT2 in lipid accumulation has been demonstrated in adipocytes, However, little is known about the effect of DGAT2 on the FA composition of these cells. Methods To investigate the role of DGAT2 in regulating lipid accumulation, FA composition and the expression of adipogenic genes, we cloned the open reading frame of the porcine DGAT2 gene and established 3T3-L1 cells that overexpressed DGAT2. Cells were then cultured in differentiation medium (DM) without FA, with a mixture of FAs (FA-DM), or containing a 13C stable isotope-labeled FA mixture (IFA-DM). The FA composition of adipocytes was analyzed by gas chromatography–mass spectrometry and gas chromatography-isotope ratio mass spectrometry. Quantitative PCR and western blotting were employed to detect expression of adipogenic genes in 3T3-L1 adipocytes cultured with FA-DM for 12 d. Results The triacylglyceride (TAG) content was significantly higher in 3T3-L1 adipocytes overexpressing DGAT2 than in control cells. When cultured in DM or FA-DM for 12 d, cells overexpressing DGAT2 showed a higher proportion of unsaturated FAs (C16:1 and C18:1). However, when cells overexpressing DGAT2 were cultured with FA-DM for 30 min, the FA composition was almost identical to that of controls. Further, the proportion of stable isotope-labeled FAs were similar in 3T3-L1 adipocytes overexpressing DGAT2 and control cells cultured in IFA-DM for 12 d. These results collectively indicate that the higher proportion of mono-unsaturated FAs, C16:1 and C18:1, may originate from de novo FA synthesis but not from the uptake of specific FAs from the medium. This hypothesis is further supported by evidence that both mRNA and protein expression of genes involved in FA

  20. In vitro BALB/3T3 cell transformation assay of nonoxynol-9 and 1,4-dioxane.

    PubMed

    Sheu, C W; Moreland, F M; Lee, J K; Dunkel, V C

    1988-01-01

    The spermicidal surfactant nonoxynol-9 (Igepal CO-630, GAF Corp.) and a potential impurity, 1,4-dioxane, were tested in the in vitro cell transformation assay using BALB/3T3 cells. Two treatment periods, 48 hr and 13 days, were used. Nonoxynol-9, tested at levels up to 10 micrograms/ml, did not induce transformation, whereas dioxane was very active in the induction of type III foci in the cultured BALB/3T3 cells.

  1. Regulation of gene expression by tobacco product preparations in cultured human dermal fibroblasts

    SciTech Connect

    Malpass, Gloria E.; Arimilli, Subhashini; Prasad, G.L.; Howlett, Allyn C.

    2014-09-01

    Skin fibroblasts comprise the first barrier of defense against wounds, and tobacco products directly contact the oral cavity. Cultured human dermal fibroblasts were exposed to smokeless tobacco extract (STE), total particulate matter (TPM) from tobacco smoke, or nicotine at concentrations comparable to those found in these extracts for 1 h or 5 h. Differences were identified in pathway-specific genes between treatments and vehicle using qRT-PCR. At 1 h, IL1α was suppressed significantly by TPM and less significantly by STE. Neither FOS nor JUN was suppressed at 1 h by tobacco products. IL8, TNFα, VCAM1, and NFκB1 were suppressed after 5 h with STE, whereas only TNFα and NFκB1 were suppressed by TPM. At 1 h with TPM, secreted levels of IL10 and TNFα were increased. Potentially confounding effects of nicotine were exemplified by genes such as ATF3 (5 h), which was increased by nicotine but suppressed by other components of STE. Within 2 h, TPM stimulated nitric oxide production, and both STE and TPM increased reactive oxygen species. The biological significance of these findings and utilization of the gene expression changes reported herein regarding effects of the tobacco product preparations on dermal fibroblasts will require additional research. - Highlights: • Tobacco product preparations (TPPs) alter gene expression in dermal fibroblasts. • Some immediate early genes critical to the inflammatory process are affected. • Different TPPs produce differential responses in certain pro-inflammatory genes.

  2. Expression and phosphorylation of delta-CaM kinase II in cultured Alzheimer fibroblasts.

    PubMed

    Cavazzin, Chiara; Bonvicini, Cristian; Nocera, Annachiara; Racchi, Marco; Kasahara, Jiro; Tardito, Daniela; Gennarelli, Massimo; Govoni, Stefano; Racagni, Giorgio; Popoli, Maurizio

    2004-10-01

    Dysregulation of calcium homeostasis is among the major cellular alterations in Alzheimer's disease (AD). We studied Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II), one of the major effectors regulating neuronal responses to changes in calcium fluxes, in cultured skin fibroblasts from subjects with sporadic AD. We found, by using PCR and Western analysis, that human fibroblasts express the delta-isoform of this kinase, and that CaM kinase II is the major Ca(2+)/calmodulin-dependent kinase in these cells. Protein expression level of the kinase was not significantly different in AD fibroblasts. However, the total activity of the kinase (stimulated by Ca(2+)/calmodulin) was significantly reduced in AD cell lines, whereas Ca(2+)-independent activity was significantly enhanced. The percent autonomy of the kinase (%Ca(2+)-independent/Ca(2+)-dependent activity) in AD cell lines was 62.8%, three-fold the corresponding percentage in control fibroblasts. The abnormal calcium-independent activity was not due to enhanced basal autophosphorylation of Thr(287). The observed abnormalities, if present in brain tissue, may be implicated either in dysfunction of neuroplasticity and cognitive functions or in dysregulation of cell cycle.

  3. Protein turnover in 3T3 cells transformed with the oncogene c-H-ras1.

    PubMed Central

    Gunn, J M; James, G

    1992-01-01

    We have examined protein turnover, growth, DNA synthesis and proliferation in three independent clones of 3T3-NR6 cells transformed with the oncogene c-H-ras1. We find that, firstly, the half-maximum concentration of serum and insulin regulating protein turnover in ras-transformed cells is significantly reduced from 0.5 to 0.3% for serum and from 4 nM to 0.5 nM for insulin, and, secondly, ras-transformed cells consistently have lower rates of protein degradation. The catabolic effect of conditioned medium or serum withdrawal is attenuated in transformed lines by maintaining lower basal rates of protein breakdown and higher basal rates of DNA and protein synthesis. Serum stimulation of growth in transformed cells is achieved in the short term by lower rates of protein breakdown rather than higher rates of protein synthesis: rates of protein synthesis become significantly higher 24 h after serum stimulation. Therefore transformed cells have higher rates of proliferation and grow to higher densities, but display characteristics common to normal cells because rates of protein synthesis decrease and protein degradation increase as a function of cell density. We conclude that higher basal rates of protein synthesis and growth with retention of the normal proliferative response to serum result from the pleiotropic nature of ras transformation, whereas lower rates of protein degradation and increased sensitivity to serum and insulin imply a direct regulatory role for ras. PMID:1575687

  4. 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.

  5. Platelet-derived growth factor (PDGF) stimulates glycogen synthase activity in 3T3 cells

    SciTech Connect

    Chan, C.P.; Bowen-Pope, D.F.; Ross, R.; Krebs, E.G.

    1986-05-01

    Hormonal regulation of glycogen synthase, an enzyme that can be phosphorylated on multiple sites, is often associated with changes in its phosphorylation state. Enzyme activation is conventionally monitored by determining the synthase activity ratio ((activity in the absence of glucose 6-P)/(activity in the presence of glucose 6-P)). Insulin causes an activation of glycogen synthase with a concomitant decrease in its phosphate content. In a previous report, the authors showed that epidermal growth factor (EGF) increases the glycogen synthase activity ratio in Swiss 3T3 cells. The time and dose-dependency of this response was similar to that of insulin. Their recent results indicate that PDGF also stimulates glycogen synthase activity. Enzyme activation was maximal after 30 min. of incubation with PDGF; the time course observed was very similar to that with insulin and EGF. At 1 ng/ml (0.03nM), PDGF caused a maximal stimulation of 4-fold in synthase activity ratio. Half-maximal stimulation was observed at 0.2 ng/ml (6 pM). The time course of changes in enzyme activity ratio closely followed that of /sup 125/I-PDGF binding. The authors data suggest that PDGF, as well as EFG and insulin, may be important in regulating glycogen synthesis through phosphorylation/dephosphorylation mechanisms.

  6. Modulation of Osteogenesis in MC3T3-E1 Cells by Different Frequency Electrical Stimulation

    PubMed Central

    Wang, Yu; Cui, Haitao; Wu, Zhenxu; Wu, Naipeng; Wang, Zongliang; Chen, Xuesi; Wei, Yen; Zhang, Peibiao

    2016-01-01

    Electrical stimulation (ES) is therapeutic to many bone diseases, from promoting fracture regeneration to orthopedic intervention. The application of ES offers substantial therapeutic potential, while optimal ES parameters and the underlying mechanisms responsible for the positive clinical impact are poorly understood. In this study, we assembled an ES cell culture and monitoring device. Mc-3T3-E1 cells were subjected to different frequency to investigate the effect of osteogenesis. Cell proliferation, DNA synthesis, the mRNA levels of osteosis-related genes, the activity of alkaline phosphatase (ALP), and intracellular concentration of Ca2+ were thoroughly evaluated. We found that 100 Hz could up-regulate the mRNA levels of collagen I, collagen II and Runx2. On the contrary, ES could down-regulate the mRNA levels of osteopontin (OPN). ALP activity assay and Fast Blue RR salt stain showed that 100 Hz could accelerate cells differentiation. Compared to the control group, 100 Hz could promote cell proliferation. Furthermore, 1 Hz to 10 Hz could improve calcium deposition in the intracellular matrix. Overall, these results indicate that 100Hz ES exhibits superior potentialities in osteogenesis, which should be beneficial for the clinical applications of ES for the treatment of bone diseases. PMID:27149625

  7. Transformation of NIH 3T3 cells with cloned fragments of human cytomegalovirus strain AD169.

    PubMed Central

    Nelson, J A; Fleckenstein, B; Galloway, D A; McDougall, J K

    1982-01-01

    NIH 3T3 cells were transfected with restriction endonuclease and cloned human cytomegalovirus DNA fragments to identify the transforming region(s). Cleavage of human cytomegalovirus strain AD169 DNA with XbaI and HindIII left a transforming region intact whereas EcoRI inactivated this function. Transfection of cells with cosmids containing human cytomegalovirus DNA spanning the entire genome resulted in transformation by one cosmid, pCM1058, with the AD169 HindIII DNA fragments E, R, T, and a'. Cells were selected for their growth in 1.2% methylcellulose. The clones isolated had a significant replating efficiency and were oncogenic in BALB/c nu/nu mice. Transfection of cosmids and plasmids containing subsets of the viral sequences in pCM1058 identified a common region possessed by all of the transforming recombinant molecules. This region was in the HindIII E fragment with the left boundary defined by the EcoRI d-R junction and the right boundary defined by the HindIII E-T junction. Further mapping and transfection experiments determined that the transforming region was contained without a 2.9-kilobase fragment between map units 0.123 and 0.14 on the prototype molecule of the AD169 strain. Images PMID:6287019

  8. 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.

  9. Microarray analysis of altered gene expression in murine fibroblasts transformed by nickel(II) to nickel(II)-resistant malignant phenotype

    SciTech Connect

    Kowara, Renata . E-mail: Renata.Kowara@nrc-cnrc.gc.ca; Karaczyn, Aldona; Cheng, Robert Y.S.; Salnikow, Konstantin; Kasprzak, Kazimierz S.

    2005-05-15

    B200 cells are Ni(II)-transformed mouse BALB/c-3T3 fibroblasts displaying a malignant phenotype and increased resistance to Ni(II) toxicity. In an attempt to find genes whose expression has been altered by the transformation, the Atlas Mouse Stress/Toxicology cDNA Expression Array (Clontech Laboratories, Inc., Palo Alto, CA) was used to analyze the levels of gene expression in both parental and Ni(II)-transformed cells. Comparison of the results revealed a significant up- or downregulation of the expression of 62 of the 588 genes present in the array (approximately 10.5%) in B200 cells. These genes were assigned to different functional groups, including transcription factors and oncogenes (9/14; fractions in parentheses denote the number of up-regulated versus the total number of genes assigned to this group), stress and DNA damage response genes (11/12), growth factors and hormone receptors (6/9), metabolism (7/7), cell adhesion (2/7), cell cycle (3/6), apoptosis (3/4), and cell proliferation (2/3). Among those genes, overexpression of beta-catenin and its downstream targets c-myc and cyclin D1, together with upregulated cyclin G, points at the malignant character of B200 cells. While the increased expression of glutathione (GSH) synthetase, glutathione-S-transferase A4 (GSTA4), and glutathione-S-transferase theta (GSTT), together with high level of several genes responding to oxidative stress, suggests the enforcement of antioxidant defenses in Ni-transformed cells.

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

    PubMed Central

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

    2013-01-01

    This study examined the anti-obesity effect and mechanism of action of blueberry peel extracts (BPE) in 3T3-L1 cells and high-fat diet (HFD)-induced obese rats. The levels of lipid accumulation were measured, along with the changes in the expression of genes and proteins associated with adipocyte differentiation in 3T3-L1 cells. Evidenced by Oil-red O staining and triglyceride assay, BPE dose-dependently inhibited lipid accumulation at concentrations of 0, 50, and 200 µg/ml. BPE decreased the expression of the key adipocyte differentiation regulator C/EBPβ, as well as the C/EBPα and PPARγ genes, during the differentiation of preadipocytes into adipocytes. Moreover, BPE down-regulated adipocyte-specific genes such as aP2 and FAS compared with control adipocytes. The specific mechanism mediating the effects of BP revealed that insulin-stimulated phosphorylation of Akt was strongly decreased, and its downstream substrate, phospho-GSK3β, was downregulated by BPE treatment in 3T3-L1 cells. Together, these data indicated that BP exerted anti-adipogenic activity by inhibiting the expression of PPARγ and C/EBPβ and the Akt signaling pathway in 3T3-L1 adipocytes. Next, we investigated whether BP extracts attenuated HFD-induced obesity in rats. Oral administration of BPE reduced HFD-induced body weight gain significantly without affecting food intake. The epididymal or perirenal adipose tissue weights were lower in rats on an HFD plus BPE compared with the tissue weights of HFD-induced obese rats. Total cholesterol and triglyceride levels in the rats fed BPE were modestly reduced, and the HDL-cholesterol level was significantly increased in HFD plus BP-fed rats compared with those of HFD-fed rats. Taken together, these results demonstrated an inhibitory effect of BP on adipogenesis through the down-regulation of C/EBPβ, C/EBPα, and PPARγ and the reduction of the phospho-Akt adipogenic factor in 3T3-L1 cells. Moreover, BPE reduced body weight gain and inhibited fat

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

    PubMed

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

    2010-12-01

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

  12. Enzyme-treated Ecklonia cava extract inhibits adipogenesis through the downregulation of C/EBPα in 3T3-L1 adipocytes

    PubMed Central

    Kim, In-Hye; Nam, Taek-Jeong

    2017-01-01

    In this study, we examined the inhibitory effects of enzyme-treated Ecklonia cava (EEc) extract on the adipogenesis of 3T3-L1 adipocytes. The components of Ecklonia cava (E. cava) were first separated and purified using the digestive enzymes pectinase (Rapidase® X-Press L) and cellulase (Rohament® CL). We found that the EEc extract contained three distinct phlorotannins: eckol, dieckol and phlorofucofuroeckol-A. Among the phlorotannins, dieckol was the most abundant in the EEc extract at 16 mg/g. Then we examined the inhibitory effects of EEc extract treatment on differentiation-related transcription factors and on adipogenesis-related gene expression in vitro using 3T3-L1 adipocytes. 3T3-L1 pre-adipocytes were used to determine the concentrations of the EEc extract and Garcinia cambogia (Gar) extract that did not result in cytotoxicity. Glucose utilization and triglyceride (TG) accumulation in the EEc-treated adipocytes were similarly inhibited by 50 µg/ml EEc and 200 µg/ml Gar, and these results were confirmed by Oil Red O staining. Protein expression of adipogenesis differentiation-related transcription factors following treatment with the EEc extract was also examined. Only the expression of CCAAT/enhancer-binding protein (C/EBP)α was decreased, while there was no effect on the expression of C/EBPβ, C/EBPδ, and peroxisome proliferator-activated receptor γ (PPARγ). Treatment with the EEc extract decreased the expression levels of adipogenesis-related genes, in particular sterol regulatory element binding protein-1c (SREBP-1c), adipocyte fatty acid binding protein (A-FABP), fatty acid synthase (FAS) and adiponectin. These results suggest that EEc extract treatment has an inhibitory effect on adipogenesis, specifically by affecting the activation of the C/EBPα signaling pathway and the resulting adipogenesis-related gene expression. PMID:28204815

  13. 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 μmol/l) markedly inhibited MC3T3-E1 cell proliferation, but this inhibitory effect was attenuated by mSGLXD. Furthermore, mSGLXD increased MC3T3-E1 cell mineralization following β-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

  14. Changes in gene expression of matrix constituents with respect to passage of ligament and tendon fibroblasts.

    PubMed

    Almarza, Alejandro J; Augustine, Serena M; Woo, Savio L-Y

    2008-12-01

    Trauma to the knee joint often results in injury to one or more supporting soft tissue structures, such as the medial collateral (MCL) and anterior cruciate (ACL) ligaments. Also, a portion of the patellar tendon (PT) is frequently used as a replacement graft for the ACL, resulting in a PT defect. The healing responses of these tissues are dramatically different and range from spontaneous healing to little or no healing. Studies have suggested that native cell behavior could be responsible for differences in healing potential. However, it is difficult to make comparisons as the reported results are based on different cellular passages which could have a dramatic effect on their potential to form healing tissues. Therefore, the objective of this study was to quantify the gene expression of collagen and other matrix constituents of fibroblasts from the MCL, ACL, and PT to document how they change with cell passage. We hypothesized that MCL fibroblasts would possess higher potential for matrix production through passages than ACL and PT cells because the MCL mounts a robust healing response unlike the ACL and PT. These differences in matrix expression would be dependent on passage because at earlier passages all cells would mostly be proliferating while at later passages they would tend to become senescent. Cells were isolated from the MCL, ACL, and PT of three rats and passaged a total of five times (Passage 1 to Passage 5). Using real time RT-PCR, expression of all genes of interest (Collagen Type I (ligament/tendon's main matrix constituent), Collagen Type III, Fibronectin, Metalloprotease-13 [MMP-13], and Tissue Inhibitor of Metallopreotease-1 [TIMP-1]) were quantitatively assessed. It was found that cell number for all three fibroblast types remained high from Passage 1 to Passage 5. There was a statistically significant increase in Collagen Type I of rat MCL fibroblasts throughout passage (p < 0.05). This was evident in the higher relative abundance (to GAPDH

  15. PAPSS2 Promotes Alkaline Phosphates Activity and Mineralization of Osteoblastic MC3T3-E1 Cells by Crosstalk and Smads Signal Pathways

    PubMed Central

    Wang, Weizhuo; Li, Fang; Wang, Kunzheng; Cheng, Bin; Guo, Xiong

    2012-01-01

    Several studies have indicated that PAPSS2 (3′-phosphoadenosine-5′-phosphosulfate synthetase 2) activity is important to normal skeletal development. Mouse PAPSS2 is predominantly expressed during the formation of the skeleton and cartilaginous elements of the mouse embryo and in newborn mice. However, the role and mechanism of PAPSS2 in bone formation remains largely unidentified. By analyzing the expression pattern of the PAPSS2 gene, we have found that PAPSS2 is expressed in bone tissue and bone formation. PAPSS2 transcripts increase during osteoblast differentiation and are in less level in RANKL-induced osteoclast like cells. By using lentivirus-mediated RNA interference (RNAi) technology, we knocked down PAPSS2 expression in MC3T3-E1 osteoblast. Silencing of PAPSS2 expression significantly decreases ALP activity and cell mineralization, inhibits expression of osteoblast marker osteopontin (OPN) and collagen I. Conversely, overexpression of PAPSS2 promotes the MC3T3-E1 to differentiate into osteoblast and mineralization. Moreover, compared to that in the control cells, the mRNA level and protein expression of phosphorylated Smad 2/3, which is a key transcriptional factor in the Smad osteoblast differentiation pathway, showed significant decreases in PAPSS2-silenced cells and increases in PAPSS2-overexpression cells. These results suggest that PAPSS2 might regulate osteoblast ALP activity and cell mineralization, probably through Smads signal pathways. PMID:22916269

  16. Insulin stimulates actin comet tails on intracellular GLUT4-containing compartments in differentiated 3T3L1 adipocytes.

    PubMed

    Kanzaki, M; Watson, R T; Khan, A H; Pessin, J E

    2001-12-28

    Incubation of isolated GLUT4-containing vesicles with Xenopus oocyte extracts resulted in a guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) and sodium orthovanadate stimulation of actin comet tails. The in vitro actin-based GLUT4 vesicle motility was inhibited by both latrunculin B and a dominant-interfering N-WASP mutant, N-WASP/Delta VCA. Preparations of gently sheared (broken) 3T3L1 adipocytes also displayed GTP gamma S and sodium orthovanadate stimulation of actin comet tails on GLUT4 intracellular compartments. Furthermore, insulin pretreatment of intact adipocytes prior to gently shearing also resulted in a marked increase in actin polymerization and actin comet tailing on GLUT4 vesicles. In addition, the insulin stimulation of actin comet tails was completely inhibited by Clostridum difficile toxin B, demonstrating a specific role for a Rho family member small GTP-binding protein. Expression of N-WASP/Delta VCA in intact cells had little effect on adipocyte cortical actin but partially inhibited insulin-stimulated GLUT4 translocation. Taken together, these data demonstrate that insulin can induce GLUT4 vesicle actin comet tails that are necessary for the efficient translocation of GLUT4 from intracellular storage sites to the plasma membrane.

  17. A novel IRS-1-associated protein, DGKζ regulates GLUT4 translocation in 3T3-L1 adipocytes.

    PubMed

    Liu, TingYu; Yu, BuChin; Kakino, Mamoru; Fujimoto, Hitoshi; Ando, Yasutoshi; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

    2016-10-14

    Insulin receptor substrates (IRSs) are major targets of insulin receptor tyrosine kinases. Here we identified diacylglycerol kinase zeta (DGKζ) as an IRS-1-associated protein, and examined roles of DGKζ in glucose transporter 4 (GLUT4) translocation to the plasma membrane. When DGKζ was knocked-down in 3T3-L1 adipocytes, insulin-induced GLUT4 translocation was inhibited without affecting other mediators of insulin-dependent signaling. Similarly, knockdown of phosphatidylinositol 4-phosphate 5-kinase 1α (PIP5K1α), which had been reported to interact with DGKζ, also inhibited insulin-induced GLUT4 translocation. Moreover, DGKζ interacted with IRS-1 without insulin stimulation, but insulin stimulation decreased this interaction. Over-expression of sDGKζ (short-form DGKζ), which competed out DGKζ from IRS-1, enhanced GLUT4 translocation without insulin stimulation. Taking these results together with the data showing that cellular PIP5K activity was correlated with GLUT4 translocation ability, we concluded that IRS-1-associated DGKζ prevents GLUT4 translocation in the absence of insulin and that the DGKζ dissociated from IRS-1 by insulin stimulation enhances GLUT4 translocation through PIP5K1α activity.

  18. 13-Methylberberine, a berberine analogue with stronger anti-adipogenic effects on mouse 3T3-L1 cells

    PubMed Central

    Chow, Yit-Lai; Sogame, Mami; Sato, Fumihiko

    2016-01-01

    Lipid metabolism modulation is a main focus of metabolic syndrome research, an area in which many natural and synthetic chemicals are constantly being screened for in vitro and in vivo activity. Berberine, a benzylisoquinoline plant alkaloid, has been extensively investigated for its anti-obesity effects and as a potential cholesterol and triglyceride-lowering drug. We screened 11 protoberberine and 2 benzophenanthridine alkaloids for their anti-adipogenic effects on 3T3-L1 adipocytes and found that 13-methylberberine exhibited the most potent activity. 13-Methylberberine down-regulated the expression of the main adipocyte differentiation transcription factors, peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT enhancer binding protein alpha (C/EBPα), as well as their target genes. PPARγ, C/EBPα, and sterol regulatory element binding protein 1 (SREBP-1) protein levels were reduced, and this lipid-reducing effect was attenuated by an AMP-activated protein kinase (AMPK) inhibitor, indicating that the effect of this compound requires the AMPK signaling pathway. Decreased Akt phosphorylation suggested reduced de novo lipid synthesis. C-13 methyl substitution of berberine increased its accumulation in treated cells, suggesting that 1