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Sample records for 3t3 l1 cells

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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 ...

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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.

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

    PubMed

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

    2012-08-01

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

  13. 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

  14. 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

  15. 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.

  16. 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.

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

    SciTech Connect

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

    2014-02-15

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

  18. 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.

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

    SciTech Connect

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

    2013-03-29

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

  20. Kaempferol and quercetin isolated from Euonymus alatus improve glucose uptake of 3T3-L1 cells without adipogenesis activity.

    PubMed

    Fang, Xian-Kang; Gao, Jie; Zhu, Dan-Ni

    2008-03-12

    Euonymus alatus as a folk medicine in China has been clinically used to treat type 2 diabetes for many years, and also exerts beneficial effects on hyperglycemia of diabetic animals. Our previous studies have isolated kaempferol and quercetin from the extract of E. alatus. In the present study, we investigated the possible mechanism of antidiabetic activity of these compounds. Kaempferol and quercetin could significantly improve insulin-stimulated glucose uptake in mature 3T3-L1 adipocytes. In addition, further experiments showed that kaempferol and quercetin served as weak partial agonists in the peroxisome proliferator-agonist receptor gamma (PPARgamma) reporter gene assay. Kaempferol and quercetin could not induce differentiation of 3T3-L1 preadipocytes as traditional PPARgamma agonist. When added together with the PPARgamma agonist rosiglitazone to 3T3-L1 preadipocytes, they could inhibit 3T3-L1 differentiation in a dose-dependent manner. Competitive ligand-binding assay confirmed that kaempferol and quercetin could compete with rosiglitazone at the same binding pocket site as PPARgamma. Kaempferol and quercetin showed significant inhibitory effects on NO production in response to lipopolysaccharide treatment in macrophage cells in which the PPARgamma was overexpressed; rosiglitazone was less potent than kaempferol and quercetin. These observations suggest that kaempferol and quercetin potentially act at multiple targets to ameliorate hyperglycemia, including by acting as partial agonists of PPARgamma.

  1. 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.

  2. Vitisin A inhibits adipocyte differentiation through cell cycle arrest in 3T3-L1 cells

    SciTech Connect

    Kim, Soon-hee; Park, Hee-Sook; Lee, Myoung-su; Cho, Yong-Jin; Kim, Young-Sup; Hwang, Jin-Taek; Sung, Mi Jeong; Kim, Myung Sunny; Kwon, Dae Young

    2008-07-18

    Inhibition of adipocyte differentiation is one approach among the anti-obesity strategies. This study demonstrates that vitisin A, a resveratrol tetramer, inhibits adipocyte differentiation most effectively of 18 stilbenes tested. Fat accumulation and PPAR{gamma} expression were decreased by vitisin A in a dose-dependent manner. Vitisin A significantly inhibited preadipocyte proliferation and consequent differentiation within the first 2 days of treatment, indicating that the anti-adipogenic effect of vitisin A was derived from anti-proliferation. Based on cell cycle analysis, vitisin A blocked the cell cycle at the G1-S phase transition, causing cells to remain in the preadipocyte state. Vitisin A increased p21 expression, while the Rb phosphorylation level was reduced. Therefore, vitisin A seems to induce G1 arrest through p21- and consequent Rb-dependent suppression of transcription. On the other hand, ERK and Akt signaling pathways were not involved in the anti-mitotic regulation by vitisin A. Taken together, these results suggest that vitisin A inhibits adipocyte differentiation through preadipocyte cell cycle arrest.

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. 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 13-methylberberine has improved absorption and higher accumulation compared to berberine. Our findings suggest that 13-methylberberine has potential as an anti-obesity drug. PMID:27917887

  12. 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.

  13. 13-Methylberberine, a berberine analogue with stronger anti-adipogenic effects on mouse 3T3-L1 cells.

    PubMed

    Chow, Yit-Lai; Sogame, Mami; Sato, Fumihiko

    2016-12-05

    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 13-methylberberine has improved absorption and higher accumulation compared to berberine. Our findings suggest that 13-methylberberine has potential as an anti-obesity drug.

  14. 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.

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

    PubMed

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

    2014-08-01

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

  16. 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.

  17. 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.

  18. 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

  19. Anti-Adipogenic Effects of Ethanol Extracts Prepared from Selected Medicinal Herbs in 3T3-L1 Cells

    PubMed Central

    Park, Min-Jun; Song, Ji-Hye; Shon, Myung-Soo; Kim, Hae Ok; Kwon, O Jun; Roh, Seong-Soo; Kim, Choon Young; Kim, Gyo-Nam

    2016-01-01

    Obesity is a major risk factor for various metabolic diseases such as cardiovascular disease, hypertension, and type 2 diabetes mellitus. In this study, we prepared ethanol extracts from Agastache rugosa (ARE), Chrysanthemum zawadskii (CZE), Mentha arvensis (MAE), Perilla frutescens (PFE), Leonurus sibiricus (LSE), Gardenia jasminoides (GJE), and Lycopus coreanus (LCE). The anti-oxidant and anti-adipogenic effects were evaluated. The IC50 values for ascorbic acid and LCE against 2,2-diphenyl-1-picrylhydrazyl radicals were 246.2 μg/mL and 166.2 μg/mL, respectively, followed by ARE (186.6 μg/mL), CZE (198.6 μg/mL), MAE (337.1 μg/mL), PFE (415.3 μg/mL), LSE (548.2 μg/mL), and GJE (626.3 μg/mL). In non-toxic concentration ranges, CZE had a strong inhibitory effect against 3T3-L1 adipogenes (84.5%) than those of the other extracts. Furthermore, the anti-adipogenic effect of CZE is largely limited in the early stage of adipogenesis, and we revealed that the inhibitory role of CZE in adipogenesis is required for the activation of Wnt signaling. Our results provide scientific evidence that the anti-adipogenic effect of CZE can be applied as an ingredient for the development of functional foods and nutri-cosmetics for obesity prevention. PMID:27752499

  20. 12-O-tetradecanoylphorbol-13-acetate disrupts actin filaments and focal contacts and enhances binding of fibronectin-coated latex beads to 3T3-L1 cells

    SciTech Connect

    Shiba, Yoshiki; Sasaki, Yasuto; Kanno, Yoshinobu )

    1988-10-01

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

  1. 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.

  2. Actions of β-apo-carotenoids in differentiating cells: differential effects in P19 cells and 3T3-L1 adipocytes.

    PubMed

    Wang, Cynthia X; Jiang, Hongfeng; Yuen, Jason J; Lee, Seung-Ah; Narayanasamy, Sureshbabu; Curley, Robert W; Harrison, Earl H; Blaner, William S

    2015-04-15

    β-Apo-carotenoids, including β-apo-13-carotenone and β-apo-14'-carotenal, are potent retinoic acid receptor (RAR) antagonists in transactivation assays. We asked how these influence RAR-dependent processes in living cells. Initially, we explored the effects of β-apo-13-carotenone and β-apo-14'-carotenal on P19 cells, a mouse embryonal carcinoma cell line that differentiates into neurons when treated with all-trans-retinoic acid. Treatment of P19 cells with either compound failed to block all-trans-retinoic acid induced differentiation. Liquid chromatography tandem mass spectrometry studies, however, established that neither of these β-apo-carotenoids accumulates in P19 cells. All-trans-retinoic acid accumulated to high levels in P19 cells. This suggests that the uptake and metabolism of β-apo-carotenoids by some cells does not involve the same processes used for retinoids and that these may be cell type specific. We also investigated the effects of two β-apo-carotenoids on 3T3-L1 adipocyte marker gene expression during adipocyte differentiation. Treatment of 3T3-L1 adipocytes with either β-apo-13-carotenone or β-apo-10'-carotenoic acid, which lacks RAR antagonist activity, stimulated adipocyte marker gene expression. Neither blocked the inhibitory effects of a relatively large dose of exogenous all-trans-retinoic acid on adipocyte differentiation. Our data suggest that in addition to acting as transcriptional antagonists, some β-apo-carotenoids act through other mechanisms to influence 3T3-L1 adipocyte differentiation.

  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. Differentiation to adipocytes in accompanied by an increase in the amounts of Gi- and Go-proteins in 3T3-L1 cells

    SciTech Connect

    Watkins, D.C.; Northup, J.K.; Malbon, C.C.

    1986-05-01

    Treatment of cultures of 3T3-L1 cells with methylisobutyl-xanthine and dexamethasone has been shown to result in accumulation of lipid and conversion to the morphology of adipocytes in more than 90% of the cells. The status of the stimulatory (Gs), inhibitory (Gi) and Go-proteins during the course of 3T3-L1 differentiation was examined. The amount of alpha subunit of Gs (..cap alpha..Gs), assayed by radiolabeling in the presence of cholera toxin and (/sup 32/P)NAD/sup +/, increased upon differentiation as previously described by others. The amounts of ..cap alpha..Gi and ..cap alpha..Go assayed by radiolabeling in the presence of pertussis toxin and (/sup 32/P)NAD/sup +/ increased 3-fold upon differentiation. Immunoblots of cell membranes subjected to gel electrophoresis in sodium dodecyl sulfate were probed with two rabbit antisera raised against bovine brain ..cap alpha..Go and with one raised against the..beta..-subunit of the bovine rod-outer-segment G-protein, referred to as transducin. The immunoblotting data confirm the increase upon differentiation of ..cap alpha..Go and also demonstrate an increase in the amount of the ..beta..-subunit. Thus differentiation of 3T3-L1 cells is accompanied by dramatic changes in the complexion of G-proteins in the membranes.

  5. 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.

  6. Shikonin suppresses ERK 1/2 phosphorylation during the early stages of adipocyte differentiation in 3T3-L1 cells

    PubMed Central

    2013-01-01

    Background The naphthoquinone pigment, shikonin, is a major component of Lithospermum erythrorhizon and has been shown to have various biological functions, including antimicrobial, anti-inflammatory, and antitumor effects. In this study, we investigated the effect of shikonin on adipocyte differentiation and its mechanism of action in 3T3-L1 cells. Methods To investigate the effects of shikonin on adipocyte differentiation, 3T3-L1 cells were induced to differentiate using 3-isobutyl-1-methylzanthine, dexamethasone, and insulin (MDI) for 8 days in the presence of 0–2 μM shikonin. Oil Red O staining was performed to determine the lipid accumulation in 3T3-L1 cells. To elucidate the anti-adipogenic mechanism of shikonin, adipogenic transcription factors, the phosphorylation levels of ERK, and adipogenic gene expression were analyzed by Western blotting and quantitative real-time PCR. To further confirm that shikonin inhibits adipogenic differentiation through downregulation of ERK 1/2 activity, 3T3-L1 cells were treated with shikonin in the presence of FGF-2, an activator, or PD98059, an inhibitor, of the ERK1/2 signaling pathway. Results Shikonin effectively suppressed adipogenesis and downregulated the protein levels of 2 major transcription factors, PPARγ and C/EBPα, as well as the adipocyte specific gene aP2 in a dose-dependent manner. qRT-PCR analysis revealed that shikonin inhibited mRNA expression of adipogenesis-related genes, such as PPARγ, C/EBPα, and aP2. Adipocyte differentiation was mediated by ERK 1/2 phosphorylation, which was confirmed by pretreatment with PD98059 (an ERK 1/2 inhibitor) or FGF-2 (an ERK 1/2 activator). The phosphorylation of ERK1/2 during the early stages of adipogenesis in 3T3-L1 cells was inhibited by shikonin. We also confirmed that FGF-2-stimulated ERK 1/2 activity was attenuated by shikonin. Conclusions These results demonstrate that shikonin inhibits adipogenic differentiation via suppression of the ERK signaling pathway

  7. 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

  8. 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

  9. 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

  10. 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.

  11. KGF and BMP-6 intervene in cellular reprogramming and in mesenchymal-epithelial transition (MET) of 3T3L1 mouse adipose cells.

    PubMed

    Reza, Abu M M T; Lee, Sungjin; Shiwani, Supriya; Singh, Naresh K

    2015-04-01

    Mesenchymal-epithelial transition (MET) is an inevitable process for cellular reprogramming. MET could be induced by suppressing epithelial-mesenchymal transition (EMT) signaling and activating an epithelial program within the cells. Aiming at MET, we investigated the potential of keratinocyte growth factor (KGF) and bone morphogenetic protein (BMP)-6 separately for the induction of MET in 3T3L1 mouse adipose cells and to trace the molecular events that probably upregulate during MET induction. KGF successfully induced MET through upregulation of epithelial related genes and transcript expression on 3T3L1 cells. In contrast, BMP-6 plays completely the reverse role through downregulation of all epithelial related genes and transcript expression. In KGF based treatment, seven genes (K8, K18, EpCAM, K5, K14, SMN1 and α-SMA) out of a total of eight genes were significantly (P < 0.05/P < 0.01) upregulated. Immunostaining and immunoblotting also revealed significant (P < 0.05/P < 0.01) expression of several epithelial-specific surface antigens and transcripts. Moreover, Ayoub Shaklar staining (specific to keratin) of KGF treated cells showed formation of keratin (reddish brown color) within cytoplasm of the cells, whereas control and BMP-6 treated cells did not. Conclusively, KGF was observed to have the potential to enhance MET and these clues could be used in future research into cellular reprogramming and regenerative medicine.

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

    PubMed Central

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

    2015-01-01

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

  13. Lignosulfonic acid promotes hypertrophy in 3T3-L1 cells without increasing lipid content and increases their 2-deoxyglucose uptake

    PubMed Central

    Hasegawa, Yasushi; Nakagawa, Erina; Kadota, Yukiya; Kawaminami, Satoshi

    2017-01-01

    Objective Adipose tissue plays a key role in the development of obesity and diabetes. We previously reported that lignosulfonic acid suppresses the rise in blood glucose levels through the inhibition of α-glucosidase activity and intestinal glucose absorption. The purpose of this study is to examine further biological activities of lignosulfonic acid. Methods In this study, we examined the effect of lignosulfonic acid on differentiation of 3T3-L1 cells. Results While lignosulfonic acid inhibited proliferation (mitotic clonal expansion) after induction of differentiation, lignosulfonic acid significantly increased the size of accumulated lipid droplets in the cells. Semi-quantitative reverse transcription polymerase chain reaction analysis showed that lignosulfonic acid increased the expression of the adipogenic transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ), leading to increased glucose transporter 4 (Glut-4) expression and 2-deoxyglucose uptake in differentiated 3T3-L1 cells. Additionally, feeding lignosulfonic acid to diabetic KK-Ay mice suppressed increase of blood glucose level. Conclusion Lignosulfonic acid may be useful as a functional anti-diabetic component of food. PMID:27383805

  14. 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.

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

    PubMed

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

    2013-02-26

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

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

    SciTech Connect

    Li, Qiang; Kamemura, Kazuo

    2014-07-18

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

  17. 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

  18. Sasa quelpaertensis Nakai extract and its constituent p-coumaric acid inhibit adipogenesis in 3T3-L1 cells through activation of the AMPK pathway.

    PubMed

    Kang, Seung-Woo; Kang, Seong-Il; Shin, Hye-Sun; Yoon, Seon-A; Kim, Jeong-Hwan; Ko, Hee-Chul; Kim, Se-Jae

    2013-09-01

    In this study, we investigated the effects of Sasa quelpaertensis Nakai extract (SQE) and its main constituent, p-coumaric acid, on adipogenesis in 3T3-L1 cells. SQE markedly inhibited adipogenesis by downregulating the expression of CCAAT/enhancer-binding protein α (C/EBPα), peroxisome proliferator-activated receptor γ (PPARγ), sterol regulatory element-binding protein-1c (SREBP-1c), and aP2. It also decreased the expression of fatty acid synthase (FAS) and adiponectin mRNAs in differentiating adipocytes. SQE increased AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation during the early phase of MDI-induced differentiation, suggesting that SQE exerted its anti-adipogenic effect via AMPK activation at an early stage of the differentiation process. p-Coumaric acid suppressed adipogenesis by attenuating the expression of C/EBPα, PPARγ, and SREBP-1c during the late phase of MDI-induced differentiation. In addition, p-coumaric acid increased the phosphorylation of AMPK and ACC, and the expression of carnitine palmitoyl transferase-1 (CPT-1) mRNA, in fully differentiated adipocytes, indicating that it promotes fatty acid β-oxidation via AMPK signaling. Taken together, our data suggest that SQE and p-coumaric acid might have the anti-obesitic effects via AMPK pathway in 3T3-L1 cells.

  19. Kaempferol suppresses lipid accumulation by inhibiting early adipogenesis in 3T3-L1 cells and zebrafish.

    PubMed

    Lee, Yeon-Joo; Choi, Hyeon-Son; Seo, Min-Jung; Jeon, Hui-Jeon; Kim, Kui-Jin; Lee, Boo-Yong

    2015-08-01

    Kaempferol is a flavonoid present in Kaempferia galanga and Opuntia ficus indica var. saboten. Recent studies have suggested that it has anti-oxidant, anti-inflammatory, anti-cancer, and anti-obesity effects. In this study, we focused on the anti-adipogenic effects of kaempferol during adipocyte differentiation. The results showed that kaempferol inhibits lipid accumulation in adipocytes and zebrafish. Oil Red O and Nile Red staining showed that the number of intracellular lipid droplets decreased in adipocytes and zebrafish treated with kaempferol. LPAATθ (lysophosphatidic acid acyltransferase), lipin1, and DGAT1 (triglyceride synthetic enzymes) and FASN and SREBP-1C (fatty acid synthetic proteins) showed decreased expression levels in the presence of kaempferol. In addition, treatment of kaempferol showed an inhibitory activity on cell cycle progression. Kaempferol delayed cell cycle progression from the S to G2/M phase through the regulation of cyclins in a dose-dependent manner. Kaempferol blocked the phosphorylation of AKT (protein kinase B) and mammalian target of rapamycin (mTOR) signaling pathway during the early stages of adipogenesis. In addition, kaempferol down-regulated pro-early adipogenic factors such as CCAAT-enhancer binding proteins β (C/EBPβ), and Krüppel-like factors (KLFs) 4 and 5, while anti-early adipogenic factors, such as KLF2 and pref-1(preadipocyte factor-1), were upregulated. These kaempferol-mediated regulations of early adipogenic factors resulted in the attenuation of late adipogenic factors such as C/EBPα and peroxisome proliferator-activated receptor γ (PPARγ). These results were supported in zebrafish based on the decrease in lipid accumulation and expression of adipogenic factors. Our results indicated that kaempferol might have an anti-obesity effect by regulating lipid metabolism.

  20. Lunasin attenuates obesity-related inflammation in RAW264.7 cells and 3T3-L1 adipocytes by inhibiting inflammatory cytokine production

    PubMed Central

    Chou, Mei-Jia; Wang, Chih-Hsuan

    2017-01-01

    Obesity has become a major threat to public health and is accompanied by chronic low-grade inflammation, which leads to various pathological developments. Lunasin, a natural seed peptide, exhibits several biological activities, such as anti-carcinogenesis, anti-inflammatory, and antioxidant activities. However, the mechanism of action of lunasin in obesity-related inflammation has not been investigated. The aim of this study was to explore whether lunasin could reduce the inflammation induced by obesity-related mediators in RAW264.7 cells and 3T3-L1 adipocytes and whether it could attenuate the crosstalk between the two cell lines. RAW264.7 cells were cultured in leptin-containing medium, adipocyte-conditioned medium (Ad-CM), or co-cultured with 3T3-L1 cells to mimic the physiology of obesity. The data showed that the secretion of pro-inflammatory cytokine interleukin-1β (IL-1β) was inhibited by lunasin after leptin activation of RAW264.7 cells. In addition, lunasin decreased monocyte chemoattractant protein-1 (MCP-1) and IL-1β secretions in the Ad-CM model. Cytokine MCP-1, IL-6, tumor necrosis factor (TNF)-α, and IL-1β secretions were significantly decreased by leptin or Ad-CM plus lipopolysaccharide stimulation. Subsequently, the co-culture of the two cells refined the direct relation between them, resulting in apparently increased MCP-1, and decreased IL-6 levels after lunasin treatment. In 3T3-L1 adipocytes, lunasin also exhibited anti-inflammatory property by inhibiting MCP-1, plasminogen activator inhibitor-1, and leptin productions stimulated by (TNF)-α, lipopolysaccharide, or RAW264.7 cell-conditioned medium. This result revealed that lunasin acts as a potential anti-inflammatory agent not only in macrophages but also in adipocytes, disrupting the crosstalk between these two cells. Therefore, this study suggests the intake of lunasin from diet or as a supplement, for auxiliary prevention or therapy in obesity-related inflammatory applications. PMID

  1. 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.

  2. Interferon-stimulated gene ISG12b1 inhibits adipogenic differentiation and mitochondrial biogenesis in 3T3-L1 cells.

    PubMed

    Li, Bing; Shin, Jonghyun; Lee, Kichoon

    2009-03-01

    Microarray analysis was performed to find a new group of genes or pathways that might be important in adipocyte development and metabolism. Among them, a mouse interferon-stimulated gene 12b1 (ISG12b1) is expressed at a 400-fold higher level in adipocytes compared with stromal-vascular cells. It is predominantly expressed in adipose tissue among other tissues we tested. Developmentally, ISG12b1 mRNA expression was initially inhibited followed by a dramatic induction during both in vivo and in vitro adipogenic differentiation. Adenovirus-mediated overexpression of ISG12b1 inhibited adipogenic differentiation in 3T3-L1 cells as shown by decreased lipid staining with Oil-Red-O and reduction in adipogenic marker proteins including peroxisome proliferator-activated receptor-gamma (PPARgamma), and CCAAT/enhancer-binding protein-alpha (C/EBPalpha). Our bioinformatics analysis for the predicted localization of ISG12b1 protein suggested the mitochondrial localization, which was confirmed by the colocalization of hemagglutinin-tagged ISG12b1 protein with mitochondrial marker MitoTracker. In addition, ISG12b1 protein was exclusively detected in protein extract from the fractionated mitochondria by Western blot analysis. Furthermore, overexpression of ISG12b1 in adipocytes reduced mitochondrial DNA content and gene expression of mitochondrial transcription factor A (mtTFA), nuclear respiratory factor 1 (NRF1), and cytochrome oxidase II, suggesting an inhibitory role of ISG12b1 in mitochondrial biogenesis and function. Activation of mitochondrial biogenesis and function by treatment with PPARgamma and PPARalpha agonists in 3T3-L1 cells and cold exposure in mice induced mitochondrial transcription factors and reduced ISG12 expression. These data demonstrated that mitochondrial-localized ISG12b1 protein inhibits adipocyte differentiation and mitochondrial biogenesis and function, implying the important role of mitochondrial function in adipocyte development and associated

  3. 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.

  4. 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.

  5. The edible red alga, Gracilaria verrucosa, inhibits lipid accumulation and ROS production, but improves glucose uptake in 3T3-L1 cells.

    PubMed

    Woo, Mi-Seon; Choi, Hyeon-Son; Lee, Ok-Hwan; Lee, Boo-Yong

    2013-07-01

    Gracilaria verrucosa is a red alga that is widely distributed in seaside areas of many countries. We examined the effect of G. verrucosa extract on adipogenesis, reactive oxygen species (ROS) production, and glucose uptake in 3T3-L1 cells. Oil red O staining and a nitroblue tetrazolium assay showed that G. verrucosa extract inhibited lipid accumulation and ROS production, respectively. mRNA levels of adipogenic transcription factors, peroxisome proliferator-activated receptor gamma and CCAAT/enhancer-binding protein alpha, as well as of their target gene, adipocyte protein 2, were reduced upon treatment with G. verrucosa extract. However, G. verrucosa extract increased glucose uptake, glucose transporter-4 expression, and AMP-activated protein kinaseα (AMPKα) phosphorylation compared to the control. Our results suggest that the anti-adipogenic and insulin-sensitive effects of G. verrucosa extract can be recapitulated to activation of AMPKα.

  6. Platyphylloside Isolated From Betula platyphylla Inhibit Adipocyte Differentiation and Induce Lipolysis Via Regulating Adipokines Including PPARγ in 3T3-L1 Cells

    PubMed Central

    Lee, Mina; Sung, Sang Hyun

    2016-01-01

    Background: Obesity causes or aggravates many health problems, both independently and in association with several pathological disorders, including Type II diabetes, hypertension, atherosclerosis, and cancer. Therefore, we screened small compounds isolated from natural products for the development of anti-obesity drugs. Objective: The purpose of this study was to investigate the anti-adipogenic activities of platyphylloside, diarylheptanoid isolated from Betula platyphylla, which was selected based on the screening using 3T3-L1 cells. Materials and Methods: To evaluate the inhibition of adipocyte differentiation and lipolysis, lipid contents of BPP on were measured using Oil Red O staining in 3T3-L1 cells. The mRNA and protein expression levels of various adipokines were measured by Quantitative real-time PCR and Western blotting analysis, respectively. Results: Platyphylloside showed significant inhibitory activity on adipocyte differentiation in 3T3-L1 cells and suppressed adipocyte differentiation even in the presence of troglitazone, a PPARγ agonist. Platyphylloside might suppress adipocyte differentiation through PPARγ, C/EBPα, and SREBP1-induced adipogenesis, which is synergistically associated with downstream adipocyte-specific gene promoters such as aP2, FAS, SCD-1, LPL, and Adiponectin. In addition, platyphylloside affected lipolysis by down-regulating perilipin and HSL and up-regulating TNFα. Conclusion: Taken together, the results reveal that platyphylloside has anti-adipogenic activity and highlight its potential in the prevention and treatment of obesity. SUMMARY The extract of B. platyphylla bark and its isolate, BPP, had anti-adipogenic activity in 3T3-L1 cells via suppression of adipocyte differentiation from preadipocytes.Treatment with BPP significantly down-regulated the expression of PPARγ, C/EBP, C/EBPβ, C/EBPδ, SREBP1c, SCD-1, FAS, aP2 and LPL.BPP induced a lipolytic response in mature adipocytes via up-regulation krof TNFá and down

  7. 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.

  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. Inhibitory effects of constituents from Morus alba var. multicaulis on differentiation of 3T3-L1 cells and nitric oxide production in RAW264.7 cells.

    PubMed

    Yang, Zhi-Gang; Matsuzaki, Keiichi; Takamatsu, Satoshi; Kitanaka, Susumu

    2011-07-19

    A new arylbenzofuran, 3',5'-dihydroxy-6-methoxy-7-prenyl-2-arylbenzofuran (1), and 25 known compounds, including moracin R (2), moracin C (3), moracin O (4), moracin P (5), artoindonesianin O (6), moracin D (7), alabafuran A (8), mulberrofuran L (9), mulberrofuran Y (10), kuwanon A (11), kuwanon C (12), kuwanon T (13), morusin (14), kuwanon E (15), sanggenon F (16), betulinic acid (17), uvaol (18), ursolic acid (19), β-sitosterol (20), oxyresveratrol 2-O-β-D-glucopyranoside (21), mulberroside A (22), mulberroside B (23), 5,7-dihydroxycoumarin 7-O-β-D-glucopyranoside (24), 5,7-dihydroxycoumarin 7-O-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside (25) and adenosine (26), were isolated from Morus alba var. multicaulis Perro. (Moraceae). Their structures were determined by spectroscopic methods. The prenyl-flavonoids 11-14, 16, triterpenoids 17,18 and 20 showed significant inhibitory activity towards the differentiation of 3T3-L1 adipocytes. The arylbenzofurans 1-10 and prenyl-flavonoids 11-16 also showed significant nitric oxide (NO) production inhibitory effects in RAW264.7 cells.

  10. 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.

  11. Bisphenol-A impairs insulin action and up-regulates inflammatory pathways in human subcutaneous adipocytes and 3T3-L1 cells.

    PubMed

    Valentino, Rossella; D'Esposito, Vittoria; Passaretti, Federica; Liotti, Antonietta; Cabaro, Serena; Longo, Michele; Perruolo, Giuseppe; Oriente, Francesco; Beguinot, Francesco; Formisano, Pietro

    2013-01-01

    Current evidence indicates that chemical pollutants may interfere with the homeostatic control of nutrient metabolism, thereby contributing to the increased prevalence of metabolic disorders. Bisphenol-A (BPA) is a lipophilic compound contained in plastic which is considered a candidate for impairing energy and glucose metabolism. We have investigated the impact of low doses of BPA on adipocyte metabolic functions. Human adipocytes derived from subcutaneous adipose tissue and differentiated 3T3-L1 cells were incubated with BPA, in order to evaluate the effect on glucose utilization, insulin sensitivity and cytokine secretion. Treatment with 1 nM BPA significantly inhibited insulin-stimulated glucose utilization, without grossly interfering with adipocyte differentiation. Accordingly, mRNA levels of the adipogenic markers PPARγ and GLUT4 were unchanged upon BPA exposure. BPA treatment also impaired insulin-activated receptor phosphorylation and signaling. Moreover, adipocyte incubation with BPA was accompanied by increased release of IL-6 and IFN-γ, as assessed by multiplex ELISA assays, and by activation of JNK, STAT3 and NFkB pathways. Treatment of the cells with the JNK inhibitor SP600125 almost fully reverted BPA effect on insulin signaling and glucose utilization. In conclusion, low doses of BPA interfere with inflammatory/insulin signaling pathways, leading to impairment of adipose cell function.

  12. 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

  13. Effects of retinoic acid and hydrogen peroxide on sterol regulatory element-binding protein-1a activation during adipogenic differentiation of 3T3-L1 cells.

    PubMed

    Abd Eldaim, Mabrouk A; Okamatsu-Ogura, Yuko; Terao, Akira; Kimura, Kazuhiro

    2010-11-01

    Both retinoic acid (RA) and oxidative stress (H2O2) increased transcription and cleavage of membrane-bound sterol regulatory element-binding protein (SREBP)-1, leading to enhanced transcription of fatty acid synthase (FAS) in hepatoma cells. On the other hand, RA and H2O2 decreased and increased lipogenesis in adipocytes, respectively, although roles of SREBP-1 activation in these effects remain to be elucidated. To elucidate its involvement, we examined the activation of SREBP-la, expression of FAS genes and lipid accumulation in 3T3-L1 cells in the presence of RA and/or H2O2. RA (1 microM) treatment suppressed expression of SREBP-1a and FAS genes and lipid accumulation. H2O2 (2 microM) treatment induced increased cleavage of SREBP-1a, without affecting amounts of SREBP-1a mRNA and precursor protein, and enhanced expression of FAS gene and lipid accumulation. Increased cleavage of SREBP-1a by H2O2 was also observed even in the presence of RA. These results suggest that H2O2, enhances a cleavage of SREBP-1a precursor protein, which independently occurs with the RA suppression of SREBP-1a gene expression, and that RA itself has no role in the SREBP-1a activation in adipocytes.

  14. 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.

  15. 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

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

    SciTech Connect

    Calvo, J.C.; Rodbard, D.; Katki, A.; Chernick, S.; Yanagishita, M. )

    1991-06-15

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

  17. Resveratrol inhibits lipogenesis of 3T3-L1 and SGBS cells by inhibition of insulin signaling and mitochondrial mass increase.

    PubMed

    Li, Shuijie; Bouzar, Célia; Cottet-Rousselle, Cécile; Zagotta, Ivana; Lamarche, Frédéric; Wabitsch, Martin; Tokarska-Schlattner, Malgorzata; Fischer-Posovszky, Pamela; Schlattner, Uwe; Rousseau, Denis

    2016-06-01

    Resveratrol is attracting much interest because of its potential to decrease body weight and increase life span, influencing liver and muscle function by increasing mitochondrial mass and energy expenditure. Even though resveratrol was already shown to reduce the adipose tissue mass in animal models, its effects on mitochondrial mass and network structure in adipocytes have not yet been studied. For this purpose, we investigated the effect of resveratrol on mitochondrial mass increase and remodeling during adipogenic differentiation of two in vitro models of adipocyte biology, the murine 3T3-L1 cell line and the human SGBS cell strain. We confirm that resveratrol inhibits lipogenesis in differentiating adipocytes, both mouse and human. We further show that this is linked to inhibition of the normally observed mitochondrial mass increase and mitochondrial remodeling. At the molecular level, the anti-lipogenic effect of resveratrol seems to be mediated by a blunted expression increase and an inhibition of acetyl-CoA carboxylase (ACC). This is one of the consequences of an inhibited insulin-induced signaling via Akt, and maintained signaling via AMP-activated protein kinase. The anti-lipogenic effect of resveratrol is further modulated by expression levels of mitochondrial ATAD3, consistent with the emerging role of this protein as an important regulator of mitochondrial biogenesis and lipogenesis. Our data suggest that resveratrol acts on differentiating preadipocytes by inhibiting insulin signaling, mitochondrial biogenesis, and lipogenesis, and that resveratrol-induced reduction of mitochondrial biogenesis and lipid storage contribute to adipose tissue weight loss in animals and humans.

  18. Evaluation of antioxidant properties of major dietary polyphenols and their protective effect on 3T3-L1 preadipocytes and red blood cells exposed to oxidative stress.

    PubMed

    Hatia, S; Septembre-Malaterre, A; Le Sage, F; Badiou-Bénéteau, A; Baret, P; Payet, B; Lefebvre d'hellencourt, C; Gonthier, M P

    2014-04-01

    Obesity has been associated with a marked risk of metabolic diseases and requires therapeutic strategies. Changes in redox status with increased oxidative stress in adipose tissue have been linked with obesity-related disorders. Thus, the biological effect of antioxidants such as polyphenols is of high interest. We aimed to measure antioxidant capacities of 28 polyphenols representative of main dietary phenolic acids, flavonoids, stilbenes and curcuminoids. Then, 14 molecules were selected for the evaluation of their effect on 3T3-L1 preadipocytes and human red blood cells exposed to oxidative stress. Analysis of reducing and free radical-scavenging capacities of compounds revealed antioxidant properties related to their structure, with higher activities for flavonoids such as quercetin and epicatechin. Their effects on preadipocytes' viability also depended on their structure, dose and time of exposure. Interestingly, most of the compounds exhibited a protective effect on preadipocytes exposed to oxidative stress, by reversing H₂O₂-induced anti-proliferative action and reactive oxygen species production. Polyphenols also exerted an anti-inflammatory effect on preadipocytes exposed to H₂O₂ by reducing IL-6 secretion. Importantly, such antioxidant and anti-inflammatory effects were observed in co-exposition (polyphenol and prooxidant during 24 h) or pretreatment (polyphenol during 24 h, then prooxidant for 24 h) conditions. Moreover, compounds protected erythrocytes from AAPH radical-induced lysis. Finally, these results led to demonstrate that antioxidant and anti-inflammatory properties of polyphenols may depend on structure, dose, time of exposure and cell conditioning with oxidative stress. Such findings should be considered for a better understanding of polyphenols' benefits in strategies aiming to prevent obesity-related diseases.

  19. Glycerol Production from Glucose and Fructose by 3T3-L1 Cells: A Mechanism of Adipocyte Defense from Excess Substrate

    PubMed Central

    Romero, María del Mar; Sabater, David; Fernández-López, José Antonio; Remesar, Xavier; Alemany, Marià

    2015-01-01

    Cultured adipocytes (3T3-L1) produce large amounts of 3C fragments; largely lactate, depending on medium glucose levels. Increased glycolysis has been observed also in vivo in different sites of rat white adipose tissue. We investigated whether fructose can substitute glucose as source of lactate, and, especially whether the glycerol released to the medium was of lipolytic or glycolytic origin. Fructose conversion to lactate and glycerol was lower than that of glucose. The fast exhaustion of medium glucose was unrelated to significant changes in lipid storage. Fructose inhibited to a higher degree than glucose the expression of lipogenic enzymes. When both hexoses were present, the effects of fructose on gene expression prevailed over those of glucose. Adipocytes expressed fructokinase, but not aldolase b. Substantive release of glycerol accompanied lactate when fructose was the substrate. The mass of cell triacylglycerol (and its lack of change) could not justify the comparatively higher amount of glycerol released. Consequently, most of this glycerol should be derived from the glycolytic pathway, since its lipolytic origin could not be (quantitatively) sustained. Proportionally (with respect to lactate plus glycerol), more glycerol was produced from fructose than from glucose, which suggests that part of fructose was catabolized by the alternate (hepatic) fructose pathway. Earlier described adipose glycerophophatase activity may help explain the glycolytic origin of most of the glycerol. However, no gene is known for this enzyme in mammals, which suggests that this function may be carried out by one of the known phosphatases in the tissue. Break up of glycerol-3P to yield glycerol, may be a limiting factor for the synthesis of triacylglycerols through control of glycerol-3P availability. A phosphatase pathway such as that described may have a potential regulatory function, and explain the production of glycerol by adipocytes in the absence of lipolytic

  20. Glycerol Production from Glucose and Fructose by 3T3-L1 Cells: A Mechanism of Adipocyte Defense from Excess Substrate.

    PubMed

    Romero, María del Mar; Sabater, David; Fernández-López, José Antonio; Remesar, Xavier; Alemany, Marià

    2015-01-01

    Cultured adipocytes (3T3-L1) produce large amounts of 3C fragments; largely lactate, depending on medium glucose levels. Increased glycolysis has been observed also in vivo in different sites of rat white adipose tissue. We investigated whether fructose can substitute glucose as source of lactate, and, especially whether the glycerol released to the medium was of lipolytic or glycolytic origin. Fructose conversion to lactate and glycerol was lower than that of glucose. The fast exhaustion of medium glucose was unrelated to significant changes in lipid storage. Fructose inhibited to a higher degree than glucose the expression of lipogenic enzymes. When both hexoses were present, the effects of fructose on gene expression prevailed over those of glucose. Adipocytes expressed fructokinase, but not aldolase b. Substantive release of glycerol accompanied lactate when fructose was the substrate. The mass of cell triacylglycerol (and its lack of change) could not justify the comparatively higher amount of glycerol released. Consequently, most of this glycerol should be derived from the glycolytic pathway, since its lipolytic origin could not be (quantitatively) sustained. Proportionally (with respect to lactate plus glycerol), more glycerol was produced from fructose than from glucose, which suggests that part of fructose was catabolized by the alternate (hepatic) fructose pathway. Earlier described adipose glycerophophatase activity may help explain the glycolytic origin of most of the glycerol. However, no gene is known for this enzyme in mammals, which suggests that this function may be carried out by one of the known phosphatases in the tissue. Break up of glycerol-3P to yield glycerol, may be a limiting factor for the synthesis of triacylglycerols through control of glycerol-3P availability. A phosphatase pathway such as that described may have a potential regulatory function, and explain the production of glycerol by adipocytes in the absence of lipolytic

  1. 4,4'-Dichlorodiphenyltrichloroethane (DDT) and 4,4'-dichlorodiphenyldichloroethylene (DDE) promote adipogenesis in 3T3-L1 adipocyte cell culture.

    PubMed

    Kim, Jonggun; Sun, Quancai; Yue, Yiren; Yoon, Kyong Sup; Whang, Kwang-Youn; Marshall Clark, J; Park, Yeonhwa

    2016-07-01

    4,4'-Dichlorodiphenyltrichloroethane (DDT), a chlorinated hydrocarbon insecticide, was extensively used in the 1940s and 1950s. DDT is mainly metabolically converted into 4,4'-dichlorodiphenyldichloroethylene (DDE). Even though most countries banned DDT in the 1970s, due to the highly lipophilic nature and very stable characteristics, DDT and its metabolites are present ubiquitously in the environment, including food. Recently, there are publications on relationships between exposure to insecticides, including DDT and DDE, and weight gain and altered glucose homeostasis. However, there are limited reports regarding DDT or DDE and adipogenesis, thus we investigated effects of DDT and DDE on adipogenesis using 3T3-L1 adipocytes. Treatment of DDT or DDE resulted in increased lipid accumulation accompanied by increased expression of CCAAT/enhancer-binding protein α (C/EBPα), peroxisome-proliferator activated receptor-γ (PPARγ), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), adipose triglyceride lipase, and leptin. Moreover, treatment of DDT or DDE increased protein levels of C/EBPα, PPARγ, AMP-activated protein kinase-α (AMPKα), and ACC, while significant decrease of phosphorylated forms of AMPKα and ACC were observed. These finding suggest that increased lipid accumulation caused by DDT and DDE may mediate AMPKα pathway in 3T3-L1 adipocytes.

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

    PubMed

    Kim, Kui-Jin; Lee, Boo-Yong

    2012-06-01

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

  3. 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.

  4. 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.

  5. Lupenone isolated from Adenophora triphylla var. japonica extract inhibits adipogenic differentiation through the downregulation of PPARγ in 3T3-L1 cells.

    PubMed

    Ahn, Eun-Kyung; Oh, Joa Sub

    2013-05-01

    Adenophora triphylla var. japonica (Campanulaceae) is known to have anti-inflammatory and anti-tussive effects. Dysfunction of adipocytes and adipose tissue in obesity is related to various inflammatory cytokines or adipokines. In this study, we investigated whether lupenone isolated from A. triphylla var. japonica extract inhibits adipocyte differentiation and expression of adipogenic marker genes in 3T3-L1 preadipocytes. We demonstrated that lupenone resulted in a significant reduction in lipid accumulation and expression of adipogenic marker genes in a dose-dependent manner. In addition, lupenone decreased the transcriptional activity of peroxisome proliferator-activated receptor γ (PPARγ) induced by troglitazone, and we also demonstrated that lupenone suppressed the PPARγ and CCAAT-enhancer-binding protein α (C/EBPα) protein levels. These findings demonstrated that lupenone isolated from A. triphylla var. japonica extract effectively inhibited adipocyte differentiation through downregulation of related transcription factor, particularly the PPARγ gene.

  6. Dodeca-2(E),4(E)-dienoic acid isobutylamide enhances glucose uptake in 3T3-L1 cells via activation of Akt signaling.

    PubMed

    Choi, Kyeong-Mi; Kim, Wonkyun; Hong, Jin Tae; Yoo, Hwan-Soo

    2017-02-01

    Dodeca-2(E),4(E)-dienoic acid isobutylamide (DDI), an alkamide derived from the plant Echinacea purpurea, promotes adipocyte differentiation and activates peroxisome proliferator-activated receptor γ, which is associated with enhanced insulin sensitivity. In the present study, we investigated whether DDI may increase glucose uptake through activation of the insulin signaling pathway in 3T3-L1 adipocytes. DDI increased insulin-stimulated glucose uptake, and expression and translocation of glucose transporter 4 in adipocytes treated with sub-optimal levels of insulin. Additionally, DDI enhanced Akt phosphorylation, whereas phosphoinositide 3-kinase/Akt inhibitors suppressed DDI-induced glucose uptake. These results suggest that DDI may improve insulin sensitivity through the activation of Akt signaling, which leads to enhanced glucose uptake.

  7. 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

  8. 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.

  9. Effect of cortisol on caspases in the co-cultured C2C12 and 3 T3-L1 cells.

    PubMed

    Muthuraman, Pandurangan

    2014-06-01

    The present study was carried out to understand the effect of cortisol on caspase expression in the C2C12 and 3 T3-L1 cells under co-culture system. Cells were co-cultured by using transwell inserts with a 0.4-μm porous membrane to separate C2C12 and 3 T3-L1 preadipocyte cells. Each cell type was grown independently on the transwell plates. Following cell differentiation, inserts containing 3 T3-L1 cells were transferred to C2C12 plates and inserts containing C2C12 cells were transferred to 3 T3-L1 plates. A total of 10 μg/μl of cortisol was added to the medium. Following treatment of cortisol for 3 days, the cells in the lower well were harvested for analysis. Caspases such as caspase 3, caspase 7, and caspase 9 were selected for the analysis. qRT-PCR results indicated the significant increase in the mRNA expression of caspase 3, caspase 7, and caspase 9. Caspase 3, 7, and 9 activities were also increased in the mono- and co-cultured C2C12 and 3 T3-L1 cells. In addition, confocal microscopical investigation indicated that cortisol increases caspase expressions in the mono- and co-cultured C2C12 and 3 T3-L1 cells. Taking all these together, we concluded that the co-culture system reflects the exact effect of cortisol on caspase expression, which is quite distinct from one dimensional mono-cultured experiments.

  10. 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

  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. 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

  13. 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

  14. 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.

  15. Persicaria hydropiper (L.) spach and its flavonoid components, isoquercitrin and isorhamnetin, activate the Wnt/β-catenin pathway and inhibit adipocyte differentiation of 3T3-L1 cells.

    PubMed

    Lee, Soung-Hoon; Kim, Bora; Oh, Myoung Jin; Yoon, Juyong; Kim, Hyun Yi; Lee, Kye Jong; Lee, Joo Dong; Choi, Kang-Yell

    2011-11-01

    Obesity, which is related to metabolic syndrome and is associated with liver disease, represents an epidemic problem demanding effective therapeutic strategies. Evidence shows that the Wnt/β-catenin pathway is closely associated with obesity and that small molecules regulating the Wnt/β-catenin pathway can potentially control adipogenesis related to obesity. Eleven plant extracts activating the Wnt/β-catenin pathway were screened by using HEK 293-TOP cells retaining the Wnt/β-catenin signaling reporter gene. An extract of Persicaria hydropiper (L.) Spach was found to activate Wnt/β-catenin signaling. P. hydropiper is grown worldwide in temperate climates and is found widely in Southeast Asia. The P. hydropiper extract inhibited the differentiation of adipocyte 3T3-L1 cells. Isoquercitrin and isorhamnetin, constituents of P. hydropiper, also activated Wnt/β-catenin signaling and suppressed the differentiation of 3T3-L1 cells. These results indicate that isoquercitrin in P. hydropiper suppresses the adipogenesis of 3T3-L1 cells via the inhibition of Wnt/β-catenin signaling. P. hydropiper and isoquercitrin may therefore be potential therapeutic agents for obesity and its associated disorders.

  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. 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.

  18. 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.

  19. Bog bilberry (Vaccinium uliginosum L.) extract reduces cultured Hep-G2, Caco-2, and 3T3-L1 cell viability, affects cell cycle progression, and has variable effects on membrane permeability.

    PubMed

    Liu, Jia; Zhang, Wei; Jing, Hao; Popovich, David G

    2010-04-01

    Bog bilberry (Vaccinium uliginosum L.) is a blue-pigmented edible berry related to bilberry (Vaccinium myrtillus L.) and the common blueberry (Vaccinium corymbosum). The objective of this study was to investigate the effect of a bog bilberry anthocyanin extract (BBAE) on cell growth, membrane permeability, and cell cycle of 2 malignant cancer cell lines, Caco-2 and Hep-G2, and a nonmalignant murine 3T3-L1 cell line. BBAE contained 3 identified anthocyanins. The most abundant anthocyanin was cyanidin-3-glucoside (140.9 +/- 2.6 microg/mg of dry weight), followed by malvidin-3-glucoside (10.3 +/- 0.3 microg/mg) and malvidin-3-galactoside (8.1 +/- 0.4 microg/mg). Hep-G2 LC50 was calculated to be 0.563 +/- 0.04 mg/mL, Caco-2 LC50 was 0.390 +/- 0.30 mg/mL and 0.214 +/- 0.02 mg/mL for 3T3-L1 cells. LDH release, a marker of membrane permeability, was significantly increased in Hep-G2 cells and Caco-2 cells after 48 and 72 h compared to 24 h. The increase was 21% at 48 h and 57% at 72 h in Caco-2 cells and 66% and 139% in Hep-G2 cells compared to 24 h. However, 3T3-L1 cells showed an unexpected significant lower LDH activity (P < or = 0.05) after 72 h of exposure corresponding to a 21% reduction in LDH release. BBAE treatment increased sub-G1 in all 3 cell lines without influencing cells in the G2/M phase. BBAE treatment reduced the growth and increased the accumulation of sub-G1 cells in 2 malignant and 1 nonmalignant cell line; however, the effect on membrane permeability differs considerably between the malignant and nonmalignant cells and may in part be due to differences in cellular membrane composition.

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. Fisetin Suppresses Lipid Accumulation in Mouse Adipocytic 3T3-L1 Cells by Repressing GLUT4-Mediated Glucose Uptake through Inhibition of mTOR-C/EBPα Signaling.

    PubMed

    Watanabe, Marina; Hisatake, Mitsuhiro; Fujimori, Ko

    2015-05-27

    3,7,3',4'-Tetrahydroxyflavone (fisetin) is a flavonoid found in vegetables and fruits having broad biological activities. Here the effects of fisetin on adipogenesis and its regulatory mechanism in mouse adipocytic 3T3-L1 cells are studied. Fisetin inhibited the accumulation of intracellular lipids and lowered the expression of adipogenic genes such as peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein (C/EBP) α and fatty acid-binding protein 4 (aP2) during adipogenesis. Moreover, the mRNA levels of genes such as acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase involved in the fatty acid biosynthesis (lipogenesis) were reduced by the treatment with fisetin. The expression level of the glucose transporter 4 (GLUT4) gene was also decreased by fisetin, resulting in down-regulation of glucose uptake. Furthermore, fisetin inhibited the phosphorylation of the mammalian target of rapamycin (mTOR) and that of p70 ribosomal S6 kinase, a target of the mTOR complex, the inhibition of which was followed by a decreased mRNA level of the C/EBPα gene. The results obtained from a chromatin immunoprecipitation assay demonstrated that the ability of C/EBPα to bind to the GLUT4 gene promoter was reduced by the treatment with fisetin, which agreed well with those obtained when 3T3-L1 cells were allowed to differentiate into adipocytes in medium in the presence of rapamycin, an inhibitor for mTOR. These results indicate that fisetin suppressed the accumulation of intracellular lipids by inhibiting GLUT4-mediated glucose uptake through inhibition of the mTOR-C/EBPα signaling in 3T3-L1 cells.

  6. 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.

  7. 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.

  8. Curcumin represses mouse 3T3-L1 cell adipogenic differentiation via inhibiting miR-17-5p and stimulating the Wnt signalling pathway effector Tcf7l2.

    PubMed

    Tian, Lili; Song, Zhuolun; Shao, Weijuan; Du, William W; Zhao, Lisa R; Zeng, Kejing; Yang, Burton B; Jin, Tianru

    2017-01-19

    Understanding mechanisms underlying adipogenic differentiation may lead to the discovery of novel therapeutic targets for obesity. Wnt signalling pathway activation leads to repressed adipogenic differentiation while certain microRNAs may regulate pre-adipocyte proliferation and differentiation. We show here that in mouse white adipose tissue, miR-17-5p level is elevated after high fat diet consumption. miR-17-5p upregulates adipogenic differentiation, as its over-expression increased while its inhibition repressed 3T3-L1 differentiation. The Tcf7l2 gene encodes a key Wnt signalling pathway effector, and its human homologue TCF7L2 is a highly regarded diabetes risk gene. We found that Tcf7l2 is an miR-17-5p target and confirmed the repressive effect of Tcf7l2 on 3T3-L1 adipogenic differentiation. The natural plant polyphenol compound curcumin possesses the body weight lowering effect. We observed that curcumin attenuated miR-17-5p expression and stimulated Tcf7l2 expression in 3T3-L1 cells. These, along with the elevation of miR-17-5p expression in mouse epididymal fat tissue in response to high fat diet consumption, allowed us to suggest that miR-17-5p is among central switches of adipogenic differentiation. It activates adipogenesis via repressing the Wnt signalling pathway effector Tcf7l2, and its own expression is likely nutritionally regulated in health and disease.

  9. Giant Oyster Mushroom Pleurotus giganteus (Agaricomycetes) Enhances Adipocyte Differentiation and Glucose Uptake via Activation of PPARγ and Glucose Transporters 1 and 4 in 3T3-L1 Cells.

    PubMed

    Paravamsivam, Puvaneswari; Heng, Chua Kek; Malek, Sri Nurestri Abdul; Sabaratnam, Vikineswary; M, Ravishankar Ram; Kuppusamy, Umah Rani

    2016-01-01

    The edible mushroom Pleurotus giganteus was tested for its effect on adipocyte differentiation and glucose uptake activity in 3T3-L1 cells. The basidiocarps of P. giganteus were soaked in methanol to obtain a crude methanol extract and then fractionated to obtain an ethyl acetate extract. In this study, cell proliferation was measured using an MTT assay, lipid accumulation using an Oil Red O assay, and glucose uptake using a fluorescence glucose uptake assay. Gene expression was measured via real-time polymerase chain reaction analysis with TaqMan primer. Ethyl acetate extract significantly enhanced adipogenic differentiation and glucose uptake in 3T3-L1 adipocytes via the expression of sterol regulatory element-binding protein, peroxisome proliferator-activated receptor γ, and phos-phatidylinositol 3-kinase/Akt. Glucose uptake was facilitated by the highly expressed glucose transporters Glut1 and Glut4. Taken together, these results suggest that P. giganteus ethyl acetate extract has an insulin-sensitizing effect on adipocytes and has potential as an adjuvant for the management of type 2 diabetes.

  10. 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

  11. 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.

  12. Curcuma longa polyphenols improve insulin-mediated lipid accumulation and attenuate proinflammatory response of 3T3-L1 adipose cells during oxidative stress through regulation of key adipokines and antioxidant enzymes.

    PubMed

    Septembre-Malaterre, Axelle; Le Sage, Fanny; Hatia, Sarah; Catan, Aurélie; Janci, Laurent; Gonthier, Marie-Paule

    2016-07-08

    Plant polyphenols may exert beneficial action against obesity-related oxidative stress and inflammation which promote insulin resistance. This study evaluated the effect of polyphenols extracted from French Curcuma longa on 3T3-L1 adipose cells exposed to H2 O2 -mediated oxidative stress. We found that Curcuma longa extract exhibited high amounts of curcuminoids identified as curcumin, demethoxycurcumin, and bisdemethoxycurcumin, which exerted free radical-scavenging activities. Curcuma longa polyphenols improved insulin-mediated lipid accumulation and upregulated peroxisome proliferator-activated receptor-gamma gene expression and adiponectin secretion which decreased in H2 O2 -treated cells. Curcuminoids attenuated H2 O2 -enhanced production of pro-inflammatory molecules such as interleukin-6, tumor necrosis factor-alpha, monocyte chemoattractant protein-1, and nuclear factor κappa B. Moreover, they reduced intracellular levels of reactive oxygen species elevated by H2 O2 and modulated the expression of genes encoding superoxide dismutase and catalase antioxidant enzymes. Collectively, these findings highlight that Curcuma longa polyphenols protect adipose cells against oxidative stress and may improve obesity-related metabolic disorders. © 2016 BioFactors, 42(4):418-430, 2016.

  13. 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.

  14. Vomifoliol 9-O-α-arabinofuranosyl (1→6)-β-D-glucopyranoside from the leaves of Diospyros Kaki stimulates the glucose uptake in HepG2 and 3T3-L1 cells.

    PubMed

    Wang, Lan; Xu, Ming Lu; Rasmussen, Søren K; Wang, Myeong-Hyeon

    2011-07-15

    A novel α-glucosidase inhibitor, vomifoliol 9-O-α-arabinofuranosyl (1→6)-β-D-glucopyranoside, was isolated for the first time from leaves of Diospyros Kaki and its bioactivity analyzed. This inhibitor exhibited strong anti-α-glucosidase activity with an IC50 value of 170.62nM and stimulated a dose-dependent increase in the uptake of a fluorescent d-glucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), in HepG2 cells at a rate higher than that of insulin controls. It was also found to be associated with adipocyte differentiation and moderate increases in 2-NBDG uptake by 3T3-L1 cells. These findings suggest that vomifoliol 9-O-α-arabinofuranosyl (1→6)-β-D-glucopyranoside could augment peripheral glucose as an insulin-sensitizing agent against Type 2 diabetes mellitus.

  15. 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

  16. 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

  17. 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.

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

    PubMed

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

    2013-01-01

    The excessive accumulation of adipocytes contributes to the development of obesity and obesity-related diseases. The interactions of several transcription factors, such as C/EBPβ, PPARγ, C/EBPα, Nrf2, and STAT3, are required for adipogenic differentiation. Dimethylfumarate (DMF), an immune modulator and antioxidant, may function as an inhibitor of STAT3 and an activator of Nrf2. This study examined whether DMF inhibits adipogenic differentiation of 3T3-L1 preadipocytes by inhibiting STAT3 or activating Nrf2. DMF suppressed 3T3-L1 preadipocyte differentiation to mature adipocytes in a dose-dependent manner as determined by Oil Red O staining. The mRNA and protein levels of adipogenic genes, including C/EBPβ, C/EBPα, PPARγ, SREBP-1c, FAS, and aP2, were significantly lower in DMF-treated 3T3-L1 preadipocytes. Suppression of adipogenic differentiation by DMF treatment resulted primarily from inhibition of the early stages of differentiation. DMF inhibits clonal expansion during adipogenic differentiation through induction of a G1 cell cycle arrest. Additionally, DMF regulates cell cycle-related proteins, such as p21, pRb, and cyclin D. DMF treatment markedly inhibited differentiation medium-induced STAT3 phosphorylation and inhibited STAT3 transcriptional activation of a reporter construct composed of four synthetic STAT3-response elements. Moreover, inhibition of endogenous Nrf2 activity using a dominant negative Nrf2 did not abolish the DMF-induced inhibition of adipogenic differentiation of 3T3-L1 preadipocytes. In summary, DMF is a negative regulator of adipogenic differentiation based on its regulation of adipogenic transcription factors and cell cycle proteins. This negative regulation by DMF is mediated by STAT3 inhibition, but is unlikely to involve Nrf2 activation.

  19. 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.

  20. 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.

  1. 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.

  2. STAT5a promotes the transcription of mature mmu-miR-135a in 3T3-L1 cells by binding to both miR-135a-1 and miR-135a-2 promoter elements.

    PubMed

    Wei, Xiajie; Cheng, Xiaoyan; Peng, Yongdong; Zheng, Rong; Chai, Jin; Jiang, Siwen

    2016-08-01

    Despite extensive research on the role of miR-135a in biological processes, very little attention has been paid to the regulation of its transcription. We have previously reported that miR-135a suppresses 3T3-L1 preadipocyte differentiation and adipogenesis by directly targeting the adenomatous polyposis coli (APC) gene and activating the canonical Wnt/β-catenin signaling pathway, but the regulatory elements that regulate the expression of the two isoforms of miR-135a (miR-135a-1 and miR-135a-2) remain poorly understood. Here, by using deletion analysis, we predicted two binding sites (-874/-856 and -2020/-2002) for the transcription factor Signal Transducers and Activators of Transcription 5a (STAT5a) within the core promoters of miR-135a-1 and miR-135a-2 (-1128/-556 and -2264/-1773), and the subsequent site-directed mutagenesis indicated that the two STAT5a binding sites regulated the activity of the miR-135a-1 and miR-135a-2 promoters. The binding of STAT5a to the miR-135a-1/2 core promoters in vitro and in cell culture was identified by electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) assays. Overexpression and RNAi knockdown of STAT5a showed that the transcription factor regulated the endogenous miR-135a expression. Additionally, The expression time frame of STAT5a and APC indicated a potential negative feedback between them. In sum, the overall results from this study indicate that STAT5a regulates miR-135a transcription by binding to both miR-135a-1 and miR135a-2 promoter elements and the findings provide novel insights into the molecular regulatory mechanisms of miR-135a during adipogenesis.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. [8-hydroxy-dihydroberberine ameliorated insulin resistance induced by high FFA and high glucose in 3T3-L1 adipocytes].

    PubMed

    Xu, Li-jun; Lu, Fu-er; Yi, Ping; Wang, Zeng-si; Wei, Shi-chao; Chen, Guang; Dong, Hui; Zou, Xin

    2009-11-01

    The purpose of the study is to investigate the effect of 8-hydroxy-dihydroberberine on insulin resistance induced by high free fatty acid (FFA) and high glucose in 3T3-L1 adipocytes and its possible molecular mechanism. Palmic acid or glucose in combination with insulin was used to induce insulin resistance in 3T3-L1 adipocytes. 8-Hydroxy-dihydroberberine and berberine were added to the cultured medium separately, which were considered as treated group and positive control group. The rate of glucose uptake was determined by 2-deoxy-[3H]-D-glucose method. The amount of glucose consumption in the medium was measured by glucose oxidase method. Cell growth and proliferation of 3T3-L1 adipocytes were detected with Cell Counting Kit-8 (CCK-8) assay. After incubated with palmic acid for 24 hours or glucose with insulin for 18 hours, the rate of glucose transport in 3T3-L1 adipocytes was inhibited by 67% and 58%, respectively. The amount of glucose consumption in 3T3-L1 adipose cells was decreased by 41% after cells were incubated with palmic acid for 24 h. However, the above changes were reversed by pretreatment with 8-hydroxy-dihydroberberine for 24 and 48 h. Significant difference existed between groups. Insulin resistance in 3T3-L1 adipocytes, which is induced by high FFA and high glucose, could be ameliorated by 8-hydroxy-dihydroberberine.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  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. 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.

  20. 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.

  1. 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.

  2. 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

  3. Hormone and pharmaceutical regulation of ASP production in 3T3-L1 adipocytes.

    PubMed

    Gao, Ying; Gauvreau, Danny; Cianflone, Katherine

    2010-04-01

    Several studies have demonstrated increases in acylation stimulating protein (ASP), and precursor protein C3 in obesity, diabetes and dyslipidemia, however the nature of the regulation is unknown. To evaluate chronic hormonal and pharmaceutical mediated changes in ASP and potential mechanisms, 3T3-L1 adipocytes were treated with physiological concentrations of relevant hormones and drugs currently used in treatment of metabolic diseases for 48 h. Medium ASP production and C3 secretion were evaluated in relation to changes in adipocyte lipid metabolism (cellular triglyceride (TG) mass, non-esterified fatty acid (NEFA) release and real-time FA uptake). Chylomicrons increased ASP production (up to 411 +/- 133% P < 0.05), while leptin, triiodothyronine, and beta-blockers atenolol and propranolol had no effect. Dexamethasone, lovastatin, rosiglitazone and rimonabant decreased ASP production (-53 to -85%, P < 0.05), associated with a decrease in the precursor protein C3 (-37% to -65%, P < 0.01). By contrast, epinephrine, progesterone, testosterone, angiotensin II and metformin also decreased ASP (-54% to -100%, P < 0.05), but without change in precursor protein C3, suggesting a direct effect on convertase activity, possibly mediated by interference (except metformin) due to marked increases in NEFA (5.6-31-fold, increased P < 0.05). Both lovastatin and metformin induced decreases in ASP were also associated with decreased TG mass (maximal -60%, P < 0.05) and real-time FA uptake (maximum -75%, P < 0.05), suggesting a change in adipocyte differentiation status. These in vitro results are consistent with in vivo ASP profiles in subjects, and suggest that ASP may be regulated through precursor C3 availability, convertase activity and differentiation status.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. Stimulatory Effects of Cinnamon Extract (Cinnamomum cassia) during the Initiation Stage of 3T3-L1 Adipocyte Differentiation

    PubMed Central

    Lee, Sang Gil; Siaw, Joanna A.; Kang, Hye Won

    2016-01-01

    Cinnamon (Cinnamomum cassia) has an anti-diabetic effect by possibly increasing the lipid storage capacity of white adipocytes; however, this effect remains controversial. The aim of this study was to examine which stage of adipogenesis is critical for the stimulatory effect of cinnamon in adipogenesis using 3T3-L1 cells. Cells were treated with cinnamon extract during three different stages of adipogenesis. We found that genes related to adipogenesis and lipogenesis were enhanced when cinnamon extract was administered during the initiation stage of differentiation but not when administered during the preadipocyte and post stages of differentiation. At the same time, genes that were involved in the regulation of fatty acid oxidation were unexpectedly upregulated. Taken together, cinnamon may boost lipid storage in white adipocytes and increase the fatty acid oxidation capacity throughout the initiation stage of differentiation, which may be beneficial for the prevention of obesity-induced type II diabetes. PMID:28231178

  9. 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.

  10. 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

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

    PubMed Central

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

    2014-01-01

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

  13. Knockdown of LYRM1 rescues insulin resistance and mitochondrial dysfunction induced by FCCP in 3T3-L1 adipocytes.

    PubMed

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

    2014-09-01

    LYR motif-containing 1 (LYRM1) was recently discovered to be involved in adipose tissue homeostasis and obesity-associated insulin resistance. We previously demonstrated that LYRM1 overexpression might contribute to insulin resistance and mitochondrial dysfunction. Additionally, knockdown of LYRM1 enhanced insulin sensitivity and mitochondrial function in 3T3-L1 adipocytes. We investigated whether knockdown of LYRM1 in 3T3-L1 adipocytes could rescue insulin resistance and mitochondrial dysfunction induced by the cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP), a mitochondrion uncoupler, to further ascertain the mechanism by which LYRM1 is involved in obesity-associated insulin resistance. Incubation of 3T3-L1 adipocytes with 1 µM FCCP for 12 h decreased insulin-stimulated glucose uptake, reduced intracellular ATP synthesis, increased intracellular reactive oxygen species (ROS) production, impaired insulin-stimulated Glucose transporter type 4 (GLUT4) translocation, and diminished insulin-stimulated tyrosine phosphorylation of Insulin receptor substrate-1 (IRS-1) and serine phosphorylation of Protein Kinase B (Akt). Knockdown of LYRM1 restored insulin-stimulated glucose uptake, rescued intracellular ATP synthesis, reduced intracellular ROS production, restored insulin-stimulated GLUT4 translocation, and rescued insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt in FCCP-treated 3T3-L1 adipocytes. This study indicates that FCCP-induced mitochondrial dysfunction and insulin resistance are ameliorated by knockdown of LYRM1.

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. 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 3T3L1 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 3T3L1 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 3T3L1 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 3T3L1 adipocytes with 1 mmol/l TUDCA, the expression levels of these three proteins decreased. Therefore, TUDCA may inhibit TNF-α-induced lipolysis in 3T3L1 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. 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

  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. 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.

  10. Automated Image Processing for Spatially Resolved Analysis of Lipid Droplets in Cultured 3T3-L1 Adipocytes

    PubMed Central

    Sims, James Kenneth; Rohr, Brian; Miller, Eric

    2015-01-01

    Cellular hypertrophy of adipose tissue underlies many of the proposed proinflammatory mechanisms for obesity-related diseases. Adipose hypertrophy results from an accumulation of esterified lipids (triglycerides) into membrane-enclosed intracellular lipid droplets (LDs). The coupling between adipocyte metabolism and LD morphology could be exploited to investigate biochemical regulation of lipid pathways by monitoring the dynamics of LDs. This article describes an image processing method to identify LDs based on several distinctive optical and morphological characteristics of these cellular bodies as they appear under bright-field. The algorithm was developed against images of 3T3-L1 preadipocyte cultures induced to differentiate into adipocytes. We show that the calculated lipid volumes are in excellent agreement with enzymatic assay data on total intracellular triglyceride content. We also demonstrate that the image processing method can efficiently characterize the highly heterogeneous spatial distribution of LDs in a culture by showing that differentiation occurs in distinct clusters separated by regions of nearly undifferentiated cells. Prospectively, the LD detection method described in this work could be applied to time-lapse data collected with simple visible light microscopy equipment to quantitatively investigate LD dynamics. PMID:25390760

  11. 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.

  12. Parabens inhibit fatty acid amide hydrolase: A potential role in paraben-enhanced 3T3-L1 adipocyte differentiation.

    PubMed

    Kodani, Sean D; Overby, Haley B; Morisseau, Christophe; Chen, Jiangang; Zhao, Ling; Hammock, Bruce D

    2016-11-16

    Parabens are a class of small molecules that are regularly used as preservatives in a variety of personal care products. Several parabens, including butylparaben and benzylparaben, have been found to interfere with endocrine signaling and to stimulate adipocyte differentiation. We hypothesized these biological effects could be due to interference with the endocannabinoid system and identified fatty acid amide hydrolase (FAAH) as the direct molecular target of parabens. FAAH inhibition by parabens yields mixed-type and time-independent kinetics. Additionally, structure activity relationships indicate FAAH inhibition is selective for the paraben class of compounds and the more hydrophobic parabens have higher potency. Parabens enhanced 3T3-L1 adipocyte differentiation in a dose dependent fashion, different from two other FAAH inhibitors URB597 and PF622. Moreover, parabens, URB597 and PF622 all failed to enhance AEA-induced differentiation. Furthermore, rimonabant, a cannabinoid receptor 1 (CB1)-selective antagonist, did not attenuate paraben-induced adipocyte differentiation. Thus, adipogenesis mediated by parabens likely occurs through modulation of endocannabinoids, but cell differentiation is independent of direct activation of CB1 by endocannabinoids.

  13. 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

  14. 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.

  15. 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.

  16. Biological effects of THC and a lipophilic cannabis extract on normal and insulin resistant 3T3-L1 adipocytes.

    PubMed

    Gallant, M; Odei-Addo, F; Frost, C L; Levendal, R-A

    2009-10-01

    Type 2 diabetes, a chronic disease, affects about 150 million people world wide. It is characterized by insulin resistance of peripheral tissues such as liver, skeletal muscle, and fat. Insulin resistance is associated with elevated levels of tumor necrosis factor alpha (TNF-alpha), which in turn inhibits insulin receptor tyrosine kinase autophosphorylation. It has been reported that cannabis is used in the treatment of diabetes. A few reports indicate that smoking cannabis can lower blood glucose in diabetics. Delta(9)-tetrahydrocannabinol (THC) is the primary psychoactive component of cannabis. This study aimed to determine the effect of a lipophilic cannabis extract on adipogenesis, using 3T3-L1 cells, and to measure its effect on insulin sensitivity in insulin resistant adipocytes. Cells were cultured in Dulbecco's modified eagle medium (DMEM) with 10% fetal bovine serum (FBS) and differentiated over a 3 day period for all studies. In the adipogenesis studies, differentiated cells were exposed to the extract in the presence and absence of insulin. Lipid content and glucose uptake was subsequently measured. Insulin-induced glucose uptake increased, while the rate of adipogenesis decreased with increasing THC concentration. Insulin-resistance was induced using TNF-alpha, exposed to the extract and insulin-induced glucose uptake measured. Insulin-induced glucose was increased in these cells after exposure to the extract. Semiquantitative real time polymerase chain reaction (RT-PCR) was performed after ribonucleic acid (RNA) extraction to evaluate the effects of the extract on glucose transporter isotype 4 (GLUT-4), insulin receptor substrate-1 (IRS-1) and IRS-2 gene expression.

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

    PubMed

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

    2013-05-01

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

  18. Nuclear phosphoproteome analysis of 3T3-L1 preadipocyte differentiation reveals system-wide phosphorylation of transcriptional regulators.

    PubMed

    Rabiee, Atefeh; Schwämmle, Veit; Sidoli, Simone; Dai, Jie; Rogowska-Wrzesinska, Adelina; Mandrup, Susanne; Jensen, Ole N

    2017-03-01

    Adipocytes (fat cells) are important endocrine and metabolic cells critical for systemic insulin sensitivity. Both adipose excess and insufficiency are associated with adverse metabolic function. Adipogenesis is the process whereby preadipocyte precursor cells differentiate into lipid-laden mature adipocytes. This process is driven by a network of transcriptional regulators (TRs). We hypothesized that protein PTMs, in particular phosphorylation, play a major role in activating and propagating signals within TR networks upon induction of adipogenesis by extracellular stimulus. We applied MS-based quantitative proteomics and phosphoproteomics to monitor the alteration of nuclear proteins during the early stages (4 h) of preadipocyte differentiation. We identified a total of 4072 proteins including 2434 phosphorylated proteins, a majority of which were assigned as regulators of gene expression. Our results demonstrate that adipogenic stimuli increase the nuclear abundance and/or the phosphorylation levels of proteins involved in gene expression, cell organization, and oxidation-reduction pathways. Furthermore, proteins acting as negative modulators involved in negative regulation of gene expression, insulin stimulated glucose uptake, and cytoskeletal organization showed a decrease in their nuclear abundance and/or phosphorylation levels during the first 4 h of adipogenesis. Among 288 identified TRs, 49 were regulated within 4 h of adipogenic stimulation including several known and many novel potential adipogenic regulators. We created a kinase-substrate database for 3T3-L1 preadipocytes by investigating the relationship between protein kinases and protein phosphorylation sites identified in our dataset. A majority of the putative protein kinases belong to the cyclin-dependent kinase family and the mitogen-activated protein kinase family including P38 and c-Jun N-terminal kinases, suggesting that these kinases act as orchestrators of early adipogenesis.

  19. Proteomic analysis of proteins associated with lipid droplets of basal and lipolytically stimulated 3T3-L1 adipocytes.

    PubMed

    Brasaemle, Dawn L; Dolios, Georgia; Shapiro, Lawrence; Wang, Rong

    2004-11-05

    Adipocytes hold the body's major energy reserve as triacylglycerols packaged in large lipid droplets. Perilipins, the most abundant proteins on these lipid droplets, play a critical role in facilitating both triacylglycerol storage and hydrolysis. The stimulation of lipolysis by beta-adrenergic agonists triggers rapid phosphorylation of perilipin and translocation of hormone-sensitive lipase to the surfaces of lipid droplets and more gradual fragmentation and dispersion of micro-lipid droplets. Because few lipid droplet-associated proteins have been identified in adipocytes, we isolated lipid droplets from basal and lipolytically stimulated 3T3-L1 adipocytes and identified the component proteins by mass spectrometry. Structural proteins identified in both preparations include perilipin, S3-12, vimentin, and TIP47; in contrast, adipophilin, caveolin-1, and tubulin selectively localized to droplets in lipolytically stimulated cells. Lipid metabolic enzymes identified in both preparations include hormone-sensitive lipase, lanosterol synthase, NAD(P)-dependent steroid dehydrogenase-like protein, acyl-CoA synthetase, long chain family member (ACSL) 1, and CGI-58. 17-beta-Hydroxysteroid dehydrogenase, type 7, was identified only in basal preparations, whereas ACSL3 and 4 and two short-chain reductase/dehydrogenases were identified on droplets from lipolytically stimulated cells. Additionally, both preparations contained FSP27, ribophorin I, EHD2, diaphorase I, and ancient ubiquitous protein. Basal preparations contained CGI-49, whereas lipid droplets from lipolytically stimulated cells contained several Rab GTPases and tumor protein D54. A close association of mitochondria with lipid droplets was suggested by the identification of pyruvate carboxylase, prohibitin, and a subunit of ATP synthase in the preparations. Thus, adipocyte lipid droplets contain specific structural proteins as well as lipid metabolic enzymes; the structural reorganization of lipid droplets in

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. 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

  6. Chromium and vanadate combination increases insulin-induced glucose uptake by 3T3-L1 adipocytes.

    PubMed

    Brautigan, David L; Kruszewski, Allison; Wang, Hong

    2006-09-01

    Insulin activates signaling pathways in target tissues through the insulin receptor and Tyr phosphorylation of intracellular proteins. Vanadate mimics insulin and enhances its actions through inhibition of protein Tyr phosphatases. Chromium is a micronutrient that enhances insulin action to normalize blood glucose, but the mechanism is not understood. Here we show that either vanadate or chromium stimulates Tyr phosphorylation of insulin receptor in mouse 3T3-L1 adipocytes compared to insulin alone, but a combination of vanadate and chromium is not additive. Phosphorylation of MAPK or 4E-BP1 as markers for insulin signaling is stimulated by vanadate plus insulin, and chromium does not enhance the effects. Vanadate robustly activates glucose uptake by 3T3-L1 adipocytes even without added insulin and increases insulin-stimulated glucose uptake. Chromium pretreatment of adipocytes slightly enhances glucose uptake in response to insulin, but significantly increases glucose uptake above that induced by insulin plus vanadate. These data show that chromium enhances glucose uptake even when Tyr phosphorylation levels are elevated by vanadate plus insulin, suggesting separate mechanisms of action for vanadate and chromium.

  7. 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.

  8. 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

  9. Peroxynitrite activates glucose uptake in 3T3-L1 adipocytes through a PI3-K-dependent mechanism.

    PubMed

    Guzman-Grenfell, Alberto M; Garcia-Macedo, Rebeca; Gonzalez-Martinez, Marco T; Hicks, Juan Jose; Medina-Navarro, Rafael

    2005-01-01

    Peroxynitrite, the product of the reaction between *NO and O2*-, is a strong oxidant and nitrating molecule, and it has been recently consideredas a component of some important signaling pathways. Herein, we report the effect of peroxynitrite on glucose uptake in 3T3-L1 adipocytes. Peroxynitrite stimulated glucose uptake and this effect was inhibited by citochalasin B, indicating the participation of facilitated GLUT transporters. Peroxynitrite-induced glucose uptake was not related to intracellular ATP, nor to external or internal calcium, but it was inhibited by the phosphatidylinositol 3-kinase (PI3-K) inhibitor, wortmannin. Additionally, we also found that peroxynitrite did not activate the insulin receptor nor the PI3-K downstream signaling protein kinase B (PKB/Akt). The dose-dependent inhibitory action of wortmannin suggests that peroxynitrite activates glucose transport without affecting GLUT transporters translocation.

  10. Pseudoginsenoside F11, a Novel Partial PPARγ Agonist, Promotes Adiponectin Oligomerization and Secretion in 3T3-L1 Adipocytes

    PubMed Central

    Wu, Guoyu; Yi, Junyang; Liu, Ling; Wang, Pengcheng; Zhang, Zhijie

    2013-01-01

    PPARγ is a nuclear hormone receptor that functions as a master regulator of adipocyte differentiation and development. Full PPARγ agonists, such as the thiazolidinediones (TZDs), have been widely used to treat type 2 diabetes. However, they are characterized by undesirable side effects due to their strong agonist activities. Pseudoginsenoside F11 (p-F11) is an ocotillol-type ginsenoside isolated from Panax quinquefolium L. (American ginseng). In this study, we found that p-F11 activates PPARγ with modest adipogenic activity. In addition, p-F11 promotes adiponectin oligomerization and secretion in 3T3-L1 adipocytes. We also found that p-F11 inhibits obesity-linked phosphorylation of PPARγ at Ser-273 by Cdk5. Therefore, p-F11 is a novel partial PPARγ agonist, which might have the potential to be developed as a new PPARγ-targeted therapeutics for type 2 diabetes. PMID:24454336

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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}.

  17. 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.

  18. 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.

  19. Coptis chinensis alkaloids exert anti-adipogenic activity on 3T3-L1 adipocytes by downregulating C/EBP-α and PPAR-γ.

    PubMed

    Choi, Jae Sue; Kim, Ji-Hye; Ali, Md Yousof; Min, Byung-Sun; Kim, Gun-Do; Jung, Hyun Ah

    2014-10-01

    Obesity is a complex, multifactorial, and chronic disease that increases the risk for type 2 diabetes, coronary heart disease and hypertension, and has become a major worldwide health problem. Developing novel anti-obesity drugs from natural products is a promising solution to the global health problem of obesity. While screening anti-obesity potentials of natural products, the methanol extract of the rhizome of Coptis chinensis (Coptidis Rhizoma) was found to significantly inhibit adipocyte differentiation and lipid contents in 3T3-L1 cells, as assessed by Oil-Red O staining. Five known alkaloids, berberine, epiberberine, coptisine, palmatine, and magnoflorine, were isolated from the n-BuOH fraction of the methanol extract of Coptidis Rhizoma. We determined the chemical structure of these alkaloids through comparisons of published nuclear magnetic resonance (NMR) spectral data. Furthermore, we screened these alkaloids for their ability to inhibit adipogenesis over a range of concentrations (12.5-50 μM). All five Coptidis Rhizoma alkaloids significantly inhibited lipid accumulation in 3T3-L1 cells without affecting cell viability in a concentration dependent manner. In addition, the five alkaloids significantly reduced the expression levels of several adipocyte marker genes including proliferator activated receptor-γ (PPAR-γ) and CCAAT/enhancer-binding protein-α (C/EBP-α). In the present study, we found that the isolated alkaloids inhibited adipogenesis in a dose-dependent manner in 3T3-L1 cells; this inhibition was attributed to their abilities to downregulate the protein levels of the adipocyte marker proteins PPAR-γ and C/EBP-α. Thus, these results suggest that Coptidis Rhizoma extract and its isolated alkaloids may be of therapeutic interest with respect to the treatment of obesity.

  20. Heat Shock Protein Augmentation of Angelica gigas Nakai Root Hot Water Extract on Adipogenic Differentiation in Murine 3T3-L1 Preadipocytes.

    PubMed

    Lumbera, Wenchie Marie L; Dela Cruz, Joseph; Yang, Seung-Hak; Hwang, Seong Gu

    2016-03-01

    There is a high association of heat shock on the alteration of energy and lipid metabolism. The alterations associated with thermal stress are composed of gene expression changes and adaptation through biochemical responses. Previous study showed that Angelica gigas Nakai (AGN) root extract promoted adipogenic differentiation in murine 3T3-L1 preadipocytes under the normal temperature condition. However, its effect in heat shocked 3T3-L1 cells has not been established. In this study, we investigated the effect of AGN root hot water extract in the adipogenic differentiation of murine 3T3-L1 preadipocytes following heat shock and its possible mechanism of action. Thermal stress procedure was executed within the same stage of preadipocyte confluence (G0) through incubation at 42°C for one hour and then allowed to recover at normal incubation temperature of 37°C for another hour before AGN treatment for both cell viability assay and Oil Red O. Cell viability assay showed that AGN was able to dose dependently (0 to 400 μg/mL) increase cell proliferation under normal incubation temperature and also was able to prevent cytotoxicity due to heat shock accompanied by cell proliferation. Confluent preadipocytes were subjected into heat shock procedure, recovery and then AGN treatment prior to stimulation with the differentiation solution. Heat shocked preadipocytes exhibited reduced differentiation as supported by decreased amount of lipid accumulation in Oil Red O staining and triglyceride measurement. However, those heat shocked preadipocytes that then were given AGN extract showed a dose dependent increase in lipid accumulation as shown by both evaluation procedures. In line with these results, real-time polymerase chain reaction (RT-PCR) and Western blot analysis showed that AGN increased adipogenic differentiation by upregulating heat shock protection related genes and proteins together with the adipogenic markers. These findings imply the potential of AGN in heat

  1. Effect of ambrex (a herbal formulation) on oxidative stress in hyperlipidemic rats and differentiation of 3T3-L1 preadipocytes

    PubMed Central

    Devi, A. Jamuna; Ravindran, Rekha; Sankar, M.; Rajkumar, Johanna

    2014-01-01

    Background: Ambrex is a polyherbal formulation which consists of Withania somnifera, Orchis mascula, Cycas circirnalis, Shorea robusta with amber. Objective: The present study was designed to explore the potential effects of ambrex on the antioxidant status in high fat diet fed rats and to investigate the possible mechanisms focusing on the gene expression involved in adipogenesis and inflammation in 3T3-L1 cell line. Materials and Methods: Male Wistar rats were divided into four groups (n = 6); Group A received normal diet, Group B received high fat diet for 30 days, Group C and D received high fat diet for 30 days and treated with ambrex (40 mg/kg b.w) and atorvastatin (10 mg/kg b.w) for successive 15 days respectively. This study also assesses the effect of ambrex on adipogenesis in 3T3-L1 adipocytes. Results: The serum total cholesterol and triglycerides were significantly decreased in ambrex treated hyperlipidemic animals when compared to untreated animals. The activities of catalase, superoxide dismutase and reduced glutathione were significantly augmented in the serum, liver, and heart of hyperlipidemic rats treated with ambrex when compared to control. Ambrex treated rats had significant reductions in malondiadehyde levels in the serum, liver and heart compared to untreated rats. In addition, we observed that treatment with ambrex resulted in a major inhibition of pre-adipocyte differentiation of 3T3-L1 cells in vitro by suppression of peroxisome proliferator activated receptor gamma, sterol regulatory binding proteins, tumor necrosis factor-α, inducible nitricoxide synthase, leptin, and upregulation of thioredoxin 1 (TRX1) and TRX2 mRNA expression. Conclusion: Therefore, ambrex may be a potential drug for treatment of hyperlipidemia and related disorders. PMID:24914283

  2. 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.

  3. 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.

  4. Proteomic analysis of cAMP-mediated signaling during differentiation of 3 T3-L1 preadipocytes.

    PubMed

    Borkowski, Kamil; Wrzesinski, Krzysztow; Rogowska-Wrzesinska, Adelina; Audouze, Karine; Bakke, Jesse; Petersen, Rasmus Koefoed; Haj, Fawaz G; Madsen, Lise; Kristiansen, Karsten

    2014-12-01

    Initiation of adipocyte differentiation is promoted by the synergistic action of insulin/insulin-like growth factor, glucocorticoids, and agents activating cAMP-dependent signaling. The action of cAMP is mediated via PKA and Epac, where at least part of the PKA function relates to strong repression of Rho kinase activity, whereas Epac counteracts the reduction in insulin/insulin-like growth factor signaling associated with complete repression of Rho kinase activity. However, detailed knowledge of the Epac-dependent branch and the interplay with PKA is still limited. In the present study, we present a comprehensive evaluation of Epac-mediated processes and their interplay with PKA during the initiation of 3 T3-L1 preadipocyte differentiation using a combination of proteomics, molecular approaches, and bioinformatics. Proteomic analyses revealed 7 proteins specifically regulated in response to Epac activation, 4 in response to PKA activation, and 11 in response to the combined activation of Epac and PKA during the initial phase of differentiation. Network analyses indicated that the identified proteins are involved in pathways of importance for glucose metabolism, inositol metabolism, and calcium-dependent signaling thereby adding a novel facet to our understanding of cAMP-mediated potentiation of adipocyte differentiation.

  5. 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.

  6. 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

  7. A novel IRS-1-associated protein, DGKζ regulates GLUT4 translocation in 3T3-L1 adipocytes

    PubMed Central

    Liu, TingYu; Yu, BuChin; Kakino, Mamoru; Fujimoto, Hitoshi; Ando, Yasutoshi; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

    2016-01-01

    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. PMID:27739494

  8. 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.

  9. 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

  10. AICAR Attenuates TNFα-Induced Inappropriate Secretion of Monocyte Chemoattractant Protein-1 and Adiponectin in 3T3-L1 Adipocytes

    PubMed Central

    Nagahara, Keiko; Ishikawa, Takuya; Nakano, Yuya; Abe, Yoshifusa; Tanaka, Daisuke; Itabashi, Kazuo

    2016-01-01

    Aim: The increase in monocyte chemoattractant protein-1 (MCP-1) and the decrease in adiponectin production from hypertrophic adipocytes are associated with adipose tissue inflammation and its metabolic complications. The aim of this study was to determine whether 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR), an adenosine monophosphate-activated protein kinase (AMPK) activator, modulates these adipocytokine productions in tumor necrosis factor-α (TNFα)-treated adipocytes. Methods: AICAR and/or other reagents were added to the culture medium, and then, TNFα was added to fully differentiated 3T3-L1 adipocytes. The MCP-1 and adiponectin production in the culture supernatant was measured by ELISA. AMPK, phosphatidylinositol 3-kinase (PI3K), and nuclear factor-κB (NF-κB) activities were also assayed. Results: Treatment with TNFα increased MCP-1 and decreased adiponectin secretion dose-dependently in the 3T3-L1 adipocytes, and AICAR significantly inhibited these TNFα-mediated changes. Interestingly, metformin, another AMPK activator, did not have such effects on these adipocytokines. Both the AMPK and PI3K systems in the cells were significantly activated by the AICAR treatment, but the effects of AICAR on adipocytokines were not weakened by the addition of dorsomorphin, an AMPK inhibitor, or LY294002, a PI3K inhibitor. Pyrrolidine dithiocarbamate (PDTC), an NF-κB inhibitor, showed protective effects similar to those as AICAR. AICAR, but not metformin, significantly inhibited the TNFα-stimulated activation of NF-κB, and dorsomorphin did not change AICAR's effect. Conclusion: AICAR attenuates the TNFα-induced secretion of MCP-1 and adiponectin in 3T3-L1 adipocytes. The observed effects of AICAR seem to be mainly due to the inhibition of NF-κB activation rather than the activation of the AMPK pathway, at least in TNFα-treated adipocytes. PMID:27170207

  11. Soshiho-Tang Aqueous Extract Exerts Antiobesity Effects in High Fat Diet-Fed Mice and Inhibits Adipogenesis in 3T3-L1 Adipocytes

    PubMed Central

    Lee, Mee-young; Kang, Byoung-Kab

    2016-01-01

    Soshiho-tang (SST; sho-saiko-to in Japanese; xiaochaihu-tang in Chinese) has generally been used to improve liver fibrosis- and cirrhosis-related symptoms in traditional Korean medicine. Although many studies have investigated the pharmacological properties of SST, its antiobesity effect has not been elucidated. Thus, our present study was carried out to evaluate the antiobesity effect of SST using a high fat diet- (HFD) induced obese mouse model and 3T3-L1 adipose cells. C57BL/6J mice were randomly divided into four groups (n = 6/group), normal diet (ND), HFD-fed group, and HFD- and SST-fed groups (S200: 200 mg/kg of SST; S600: 600 mg/kg of SST) and given HFD with or without SST extract for 8 weeks. 3T3-L1 preadipocytes were differentiated into adipocytes for 8 days with or without SST. In the HFD-fed obese mice, body weight and fat accumulation in adipose tissue were significantly reduced by SST administration. Compared with control-differentiated adipocytes, SST significantly inhibited lipid accumulation by decreasing the triglyceride (TG) content and leptin concentration in 3T3-L1 adipocytes. SST also decreased the expression of adipogenesis-related genes including lipoprotein lipase (LPL), fatty acid binding protein 4 (FABP4), CCAAT/enhancer-binding protein-alpha (C/EBP-α), and peroxisome proliferator-activated receptor-gamma (PPAR-γ). Our findings suggest that SST has potential as a nontoxic antiobesity medication. PMID:27777595

  12. 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.

  13. Coffee extract inhibits adipogenesis in 3T3-L1 preadipocyes by interrupting insulin signaling through the downregulation of IRS1

    PubMed Central

    Maki, Chihiro; Funakoshi-Tago, Megumi; Aoyagi, Ryohei; Ueda, Fumihito; Kimura, Masaki; Kobata, Kenji; Tago, Kenji; Tamura, Hiroomi

    2017-01-01

    Although epidemiological data have indicated that a strong negative association exists between coffee consumption and the prevalence of obesity-associated diseases, the molecular mechanisms by which coffee intake prevents obesity-associated diseases has not yet been elucidated. In this study, we found that coffee intake significantly suppressed high-fat diet (HFD)-induced metabolic alternations such as increases in body weight and the accumulation of adipose tissue, and up-regulation of glucose, free fatty acid, total cholesterol and insulin levels in the blood. We also found that coffee extract significantly inhibited adipogenesis in 3T3-L1 preadipocytes. In the early phase of adipogenesis, 3T3-L1 cells treated with coffee extract displayed the retardation of cell cycle entry into the G2/M phase called as mitotic clonal expansion (MCE). Coffee extract also inhibited the activation of CCAAT/enhancer-binding protein β (C/EBPβ) by preventing its phosphorylation by ERK. Furthermore, the coffee extract suppressed the adipogenesis-related events such as MCE and C/EBPβ activation through the down-regulation of insulin receptor substrate 1 (IRS1). The stability of the IRS1 protein was markedly decreased by the treatment with coffee extract due to proteasomal degradation. These results have revealed an anti-adipogenic function for coffee intake and identified IRS1 as a novel target for coffee extract in adipogenesis. PMID:28282409

  14. Chicoric acid induces apoptosis in 3T3-L1 preadipocytes through ROS-mediated PI3K/Akt and MAPK signaling pathways.

    PubMed

    Xiao, Haifang; Wang, Jing; Yuan, Li; Xiao, Chunxia; Wang, Yutang; Liu, Xuebo

    2013-02-20

    Chicoric acid has been reported to possess various bioactivities. However, the antiobesity effects of chicoric acid remain poorly understood. In this study, we investigated the effects of chicoric acid on 3T3-L1 preadipocytes and its molecular mechanisms of apoptosis. Chicoric acid inhibited cell viability and induced apoptosis in 3T3-L1 preadipocytes which was characterized by chromatin condensation and poly ADP-ribose-polymerase (PARP) cleavage. Mitochondrial membrane potential (MMP) loss, Bax/Bcl-2 dysregulation, cytochrome c release, and caspase-3 activation were observed, indicating mitochondria-dependent apoptosis induced by chicoric acid. Furthermore, PI3K/Akt and MAPK (p38 MAPK, JNK, and ERK1/2) signaling pathways were involved in chicoric acid-induced apoptosis. The employment of protein kinase inhibitors LY294002, SB203580, SP600125, and U0126 revealed that PI3K/Akt signaling pathway interplayed with MAPK signaling pathways. Moreover, chicoric acid induced reactive oxygen species (ROS) generation. Pretreatment with the antioxidant N-acetylcysteine (NAC) significantly blocked cell death and changes of Akt and MAPK signalings induced by chicoric acid. In addition, chicoric acid down regulated HO-1 and COX-2 via the PI3K/Akt pathway.

  15. Real-time monitoring of inflammation status in 3T3-L1 adipocytes possessing a secretory Gaussia luciferase gene under the control of nuclear factor-kappa B response element

    SciTech Connect

    Nagasaki, Haruka; Yoshimura, Takeshi; Aoki, Naohito

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer Inflammation status in adipocytes can be monitored by the new assay system. Black-Right-Pointing-Pointer Only an aliquot of conditioned medium is required without cell lysis. Black-Right-Pointing-Pointer Inflammation-attenuating compounds can be screened more conveniently. -- Abstract: We have established 3T3-L1 cells possessing a secretory Gaussia luciferase (GLuc) gene under the control of nuclear factor-kappa B (NF-{kappa}B) response element. The 3T3-L1 cells named 3T3-L1-NF-{kappa}B-RE-GLuc could differentiate into adipocyte as comparably as parental 3T3-L1 cells. Inflammatory cytokines such as tumor necrosis factor (TNF)-{alpha} and interleukin (IL)-1{beta} induced GLuc secretion of 3T3-L1-NF-{kappa}B-RE-GLuc adipocytes in a concentration- and time-dependent manner. GLuc secretion of 3T3-L1-NF-{kappa}B-RE-GLuc adipocytes was also induced when cultured with RAW264.7 macrophages and was dramatically enhanced by lipopolysaccharide (LPS)-activated macrophages. An NF-{kappa}B activation inhibitor BAY-11-7085 and an antioxidant N-acetyl cysteine significantly suppressed GLuc secretion induced by macrophages. Finally, we found that rosemary-derived carnosic acid strongly suppressed GLuc secretion induced by macrophages and on the contrary up-regulated adiponectin secretion. Collectively, by using 3T3-L1-NF-{kappa}B-RE-GLuc adipocytes, inflammation status can be monitored in real time and inflammation-attenuating compounds can be screened more conveniently.

  16. Kirenol inhibits adipogenesis through activation of the Wnt/β-catenin signaling pathway in 3T3-L1 adipocytes

    SciTech Connect

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

    2014-03-07

    Highlights: • Kirenol inhibits the adipogenic transcription factors and lipogenic enzymes. • Kirenol stimulates the Wnt/β-catenin signaling pathway components. • Kirenol inhibits adipogenesis through activation of the Wnt/β-catenin signaling pathway. - Abstract: Kirenol, a natural diterpenoid compound, has been reported to possess anti-oxidant, anti-inflammatory, anti-allergic, and anti-arthritic activities; however, its anti-adipogenic effect remains to be studied. The present study evaluated the effect of kirenol on anti-adipogenesis through the activation of the Wnt/β-catenin signaling pathway. Kirenol prevented intracellular lipid accumulation by down-regulating key adipogenesis transcription factors [peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding proteins α (C/EBPα), and sterol regulatory element binding protein-1c (SREBP-1c)] and lipid biosynthesis-related enzymes [fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC)], as well as adipocytokines (adiponectin and leptin). Kirenol effectively activated the Wnt/β-catenin signaling pathway, in which kirenol up-regulated the expression of low density lipoprotein receptor related protein 6 (LRP6), disheveled 2 (DVL2), β-catenin, and cyclin D1 (CCND1), while it inactivated glycogen synthase kinase 3β (GSK3β) by increasing its phosphorylation. Kirenol down-regulated the expression levels of PPARγ and C/EBPα, which were up-regulated by siRNA knockdown of β-catenin. Overall, kirenol is capable of inhibiting the differentiation and lipogenesis of 3T3-L1 adipocytes through the activation of the Wnt/β-catenin signaling pathway, suggesting its potential as natural anti-obesity agent.

  17. Glycine suppresses TNF-α-induced activation of NF-κB in differentiated 3T3-L1 adipocytes.

    PubMed

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

    2012-08-15

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

  18. Berberine attenuates cAMP-induced lipolysis via reducing the inhibition of phosphodiesterase in 3T3-L1 adipocytes.

    PubMed

    Zhou, Libin; Wang, Xiao; Yang, Ying; Wu, Ling; Li, Fengying; Zhang, Rong; Yuan, Guoyue; Wang, Ning; Chen, Mingdao; Ning, Guang

    2011-04-01

    Berberine, a hypoglycemic agent, has been shown to decrease plasma free fatty acids (FFAs) level in insulin-resistant rats. In the present study, we explored the mechanism responsible for the antilipolytic effect of berberine in 3T3-L1 adipocytes. It was shown that berberine attenuated lipolysis induced by catecholamines, cAMP-raising agents, and a hydrolyzable cAMP analog, but not by tumor necrosis factor α and a nonhydrolyzable cAMP analog. Unlike insulin, the inhibitory effect of berberine on lipolysis in response to isoproterenol was not abrogated by wortmannin, an inhibitor of phosphatidylinositol 3-kinase, but additive to that of PD98059, an extracellular signal-regulated kinase kinase inhibitor. Prior exposure of adipocytes to berberine decreased the intracellular cAMP production induced by isoproterenol, forskolin, and 3-isobutyl-1-methylxanthine (IBMX), along with hormone-sensitive lipase (HSL) Ser-563 and Ser-660 dephosphorylation, but had no effect on perilipin phosphorylation. Berberine stimulated HSL Ser-565 as well as adenosine monophosphate-activated protein kinase (AMPK) phosphorylation. However, compound C, an AMPK inhibitor, did not reverse the regulatory effect of berberine on HSL Ser-563, Ser-660, and Ser-565 phosphorylation, nor the antilipolytic effect of berberine. Knockdown of AMPK using RNA interference also failed to restore berberine-suppressed lipolysis. cAMP-raising agents increased AMPK activity, which was not additive to that of berberine. Stimulation of adipocytes with berberine increased phosphodiesterase (PDE) 3B and PDE4 activity measured by hydrolysis of (3)[H]cAMP. These results suggest that berberine exerts an antilipolytic effect mainly by reducing the inhibition of PDE, leading to a decrease in cAMP and HSL phosphorylation independent of AMPK pathway.

  19. Germinated brown rice extract inhibits adipogenesis through the down-regulation of adipogenic genes in 3T3-L1 adipocytes.

    PubMed

    Ho, Jin-Nyoung; Son, Mi-Eun; Lim, Won-Chul; Lim, Seung-Taik; Cho, Hong-Yon

    2013-09-01

    The aim of this study was to examine the anti-adipogenic effect of germinated brown rice methanol extract (GBR) in 3T3-L1 adipocytes. The GBR inhibited adipocyte differentiation was measured by Oil Red O staining and glycerol-3-phosphate dehydrogenase (GPDH) activity in a dose-dependent manner without initiating any cytotoxicity. The mRNA levels of adipogenic transcription factors such as CCAAT/enhancer binding protein (C/EBPα), proliferator-activated receptorγ (PPARγ), and sterol regulatory element-binding protein-1c (SREBP-1c), and adipogenic genes, such as fatty acid synthase (FAS), adipocyte fatty acid-binding protein (aP2), and lipoprotein lipase (LPL), were significantly down-regulated by treatment with GBR when compared to that of untreated control cells. Moreover, tumor necrosis factor-α (TNF-α) and interlukin-6 (IL-6) mRNA expressions were attenuated by GBR in mature adipocytes. These data suggest that GBR exhibits an anti-adipogenic effect through the suppression of adipogenesis in 3T3-L1 adipocytes.

  20. Caffeine inhibits adipogenesis through modulation of mitotic clonal expansion and the AKT/GSK3 pathway in 3T3-L1 adipocytes.

    PubMed

    Kim, Ah-Reum; Yoon, Bo Kyung; Park, Hyounkyoung; Seok, Jo Woon; Choi, Hyeonjin; Yu, Jung Hwan; Choi, Yoonjeong; Song, Su Jin; Kim, Ara; Kim, Jae-Woo

    2016-02-01

    Caffeine has been proposed to have several beneficial effects on obesity and its related metabolic diseases; however, how caffeine affects adipocyte differentiation has not been elucidated. In this study, we demonstrated that caffeine suppressed 3T3-L1 adipocyte differentiation and inhibited the expression of CCAAT/enhancer binding protein (C/EBP)α and peroxisome proliferator-activated receptor (PPAR)γ, two main adipogenic transcription factors. Anti-adipogenic markers, such as preadipocyte secreted factor (Pref)-1 and Krüppel-like factor 2, remained to be expressed in the presence of caffeine. Furthermore, 3T3-L1 cells failed to undergo typical mitotic clonal expansion in the presence of caffeine. Investigation of hormonal signaling revealed that caffeine inhibited the activation of AKT and glycogen synthase kinase (GSK) 3 in a dose-dependent manner, but not extracellular signal-regulated kinase (ERK). Our data show that caffeine is an anti-adipogenic bioactive compound involved in the modulation of mitotic clonal expansion during adipocyte differentiation through the AKT/GSK3 pathway. [BMB Reports 2016; 49(2): 111-115].

  1. The v-SNARE Vti1a regulates insulin-stimulated glucose transport and Acrp30 secretion in 3T3-L1 adipocytes.

    PubMed

    Bose, Avirup; Guilherme, Adilson; Huang, Shaohui; Hubbard, Andrea C; Lane, Charles R; Soriano, Neil A; Czech, Michael P

    2005-11-04

    Regulated exocytosis in adipocytes mediates key functions, exemplified by insulin-stimulated secretion of peptides such as adiponectin and recycling of intracellular membranes containing GLUT4 glucose transporters to the cell surface. Using a proteomics approach, the v-SNARE Vti1a (vps10p tail interacting 1a) was identified by mass spectrometry in purified GLUT4-containing membranes. Insulin treatment of 3T3-L1 adipocytes decreased the amounts of both Vti1a and GLUT4 in these membranes, confirming that Vti1a is a component of insulin-sensitive GLUT4-containing vesicles. In the basal state, endogenous Vti1a colocalizes exclusively with perinuclear GLUT4. Although Vti1a has previously been reported to be a v-SNARE localized in the trans-Golgi network, treatment with brefeldin A failed to significantly modify Vti1a or GLUT4 localization while completely dispersing Golgi and trans-Golgi network marker proteins. Furthermore, depletion of Vti1a protein in cultured adipocytes through small interfering RNA-based gene silencing significantly inhibited both adiponectin secretion and insulin-stimulated deoxyglucose uptake. Taken together, these results suggest that the v-SNARE Vti1a may regulate a step common to both GLUT4 and Acrp30 trafficking in 3T3-L1 adipocytes.

  2. 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.

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

    PubMed

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

    2014-08-01

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

  4. 18O-Tracer Metabolomics Reveals Protein Turnover and CDP-Choline Cycle Activity in Differentiating 3T3-L1 Pre-Adipocytes

    PubMed Central

    Kirkwood, Jay S.; Miranda, Cristobal L.; Bobe, Gerd; Maier, Claudia S.; Stevens, Jan F.

    2016-01-01

    The differentiation of precursor cells into mature adipocytes (adipogenesis) has been an area of increased focus, spurred by a rise in obesity rates. Though our understanding of adipogenesis and its regulation at the cellular level is growing, many questions remain, especially regarding the regulation of the metabolome. The 3T3-L1 cell line is the most well characterized cellular model of adipogenesis. Using a time course metabolomics approach, we show that the 3T3-L1 preadipocyte metabolome is greatly altered during the first 48 hours of differentiation, where cells go through about two rounds of cell division, a process known as mitotic clonal expansion. Short-chain peptides were among several small molecules that were increased during mitotic clonal expansion. Additional indicators of protein turnover were also increased, including bilirubin, a degradation product of heme-containing proteins, and 3-methylhistidine, a post-translationally modified amino acid that is not reutilized for protein synthesis. To study the origin of the peptides, we treated differentiating preadipocytes with 18O labeled water and found that 18O was incorporated into the short chain peptides, confirming them, at least in part, as products of hydrolysis. Inhibitors of the proteasome or matrix metalloproteinases affected the peptide levels during differentiation, but inhibitors of autophagy or peptidases did not. 18O was also incorporated into several choline metabolites including cytidine 5'-diphosphocholine (CDP-choline), glycerophosphocholine, and several phosphatidylcholine species, indicative of phosphatidylcholine synthesis/degradation and of flux through the CDP-choline cycle, a hallmark of proliferating cells. 18O-Tracer metabolomics further showed metabolic labeling of glutamate, suggestive of glutaminolysis, also characteristic of proliferating cells. Together, these results highlight the utility of 18O isotope labeling in combination with metabolomics to uncover changes in

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

    NASA Astrophysics Data System (ADS)

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

    2012-04-01

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

  6. Trans-10,cis-12 conjugated linoleic acid (CLA) interferes with lipid droplet accumulation during 3T3-L1 preadipocyte differentiation.

    PubMed

    Yeganeh, Azadeh; Taylor, Carla G; Tworek, Leslee; Poole, Jenna; Zahradka, Peter

    2016-07-01

    In this study, we hypothesize that the biologically active isomers of conjugated linoleic acid (CLA), cis-9,trans-11 (c9,t11) and trans-10,cis-12 (t10,c12) CLA, have different effects on early and late stages 3T3-L1 preadipocyte differentiation. Both c9-t11 and t10-c12CLA stimulated early stage pre-adipocyte differentiation (day 2), while t10-c12CLA inhibited late differentiation (day 8) as determined by lipid droplet numbers and both perilipin-1 levels and phosphorylation state. At day 8, the adipokines adiponectin, chemerin and adipsin were all reduced in t10-c12CLA treated cells versus control cells. Immunofluorescence microscopy showed perilipin-1 was present solely on lipid droplets on day 8 in t10-c12 treated 3T3-L1 cells, whereas preilipin-1 was also located in the perinuclear region in control and c9-t11 treated cells. The t10-c12CLA isomer also decreased levels of hormone-sensitive lipase and inhibited lipolysis. These findings indicate that the decrease in lipid droplets caused by t10-c12CLA is the result of an inhibition of lipid droplet production during adipogenesis rather than a stimulation of lipolysis. Additionally, treatment with Gö6976 blocked the effect of t10-c12CLA on perilipin-1 phosphorylation, implicating PKCα in perilipin-1 phosphorylation, and thus a regulator of triglyceride catabolism. These data are supported by evidence that t10-c12CLA activated PKCα. These are the first data to show that CLA isomers can affect lipid droplet dynamics in adipocytes through PKCα.

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

    PubMed Central

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

    2015-01-01

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

  8. 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

  9. 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

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

    SciTech Connect

    Xue, Peng; Hou, Yongyong; Zhang, Qiang; Woods, Courtney G.; Yarborough, Kathy; Liu, Huiyu; Sun, Guifan; Andersen, Melvin E.; Pi, Jingbo

    2011-04-08

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

  11. Phlorotannins isolated from the edible brown alga Ecklonia stolonifera exert anti-adipogenic activity on 3T3-L1 adipocytes by downregulating C/EBPα and PPARγ.

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2015-05-01

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

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

    PubMed

    Lazra, Yulia; Falach, Alona; Frenkel, Lital; Rozenberg, Konstantin; Sampson, Sanford; Rosenzweig, Tovit

    2015-05-01

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

  14. Long-term ritonavir exposure increases fatty acid and glycerol recycling in 3T3-L1 adipocytes as compensatory mechanisms for increased triacylglycerol hydrolysis.

    PubMed

    Adler-Wailes, Diane C; Guiney, Evan L; Wolins, Nathan E; Yanovski, Jack A

    2010-05-01

    Lipodystrophy with high nonesterified fatty acid (FA) efflux is reported in humans receiving highly active antiretroviral therapy (HAART) to treat HIV infection. Ritonavir, a common component of HAART, alters adipocyte FA efflux, but the mechanism for this effect is not established. To investigate ritonavir-induced changes in FA flux and recycling through acylglycerols, we exposed differentiated murine 3T3-L1 adipocytes to ritonavir for 14 d. FA efflux, uptake, and incorporation into acylglycerols were measured. To identify a mediator of FA efflux, we measured adipocyte triacylglycerol lipase (ATGL) transcript and protein. To determine whether ritonavir-treated adipocytes increased glycerol backbone synthesis for FA reesterification, we measured labeled glycerol and pyruvate incorporation into triacylglycerol (TAG). Ritonavir-treated cells had increased FA efflux, uptake, and incorporation into TAG (all P < 0.01). Ritonavir increased FA efflux without consistently increasing glycerol release or changing TAG mass, suggesting increased partial TAG hydrolysis. Ritonavir-treated adipocytes expressed significantly more ATGL mRNA (P < 0.05) and protein (P < 0.05). Ritonavir increased glycerol (P < 0.01) but not pyruvate (P = 0.41), utilization for TAG backbone synthesis. Consistent with this substrate utilization, glycerol kinase transcript (required for glycerol incorporation into TAG backbone) was up-regulated (P < 0.01), whereas phosphoenolpyruvate carboxykinase transcript (required for pyruvate utilization) was down-regulated (P < 0.001). In 3T3-L1 adipocytes, long-term ritonavir exposure perturbs FA metabolism by increasing ATGL-mediated partial TAG hydrolysis, thus increasing FA efflux, and leads to compensatory increases in FA reesterification with glycerol and acylglycerols. These changes in FA metabolism may, in part, explain the increased FA efflux observed in ritonavir-associated lipodystrophy.

  15. Antiobesity effects of quercetin-rich onion peel extract on the differentiation of 3T3-L1 preadipocytes and the adipogenesis in high fat-fed rats.

    PubMed

    Moon, Jiyoung; Do, Hyun-Ju; Kim, Oh Yoen; Shin, Min-Jeong

    2013-08-01

    The aim of the present study was to examine the effect of quercetin-rich onion peel extract (OPE) on anti-differentiation in 3T3-L1 preadipocytes and the antiobesity in high-fat fed rats. We found that lipid accumulations and TG contents in 3T3-L1 cells were markedly suppressed by OPE. The mRNA levels of activating protein (AP2) were down-regulated and those of carnitine palmitoyl transferase-1 α (CPT-1α) and fatty acid binding protein 4 (FABP4) were up-regulated by 75 and 100 μg/ml OPE. Body weight, retroperitoneal and mesenteric fat weights of SD rats were significantly lower in the 8 week high fat (HF) diet+0.72% OPE group than in the HF group. Peroxisome proliferator-activated receptor (PPAR)γ mRNA levels were down-regulated in the epididymal fat of OPE than those of control and HF, and significant down-regulation of CCAAT/enhancer binding protein (C/EBP)α mRNA levels in OPE was also observed than the control. The mRNA levels of CPT-1α and uncoupling protein-1 (UCP-1) were up-regulated by the OPE, while those of fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) were down-regulated in HF and OPE groups compared to control group. These results suggest that quercentin-enriched OPE may have antiobesity effects by suppressing preadipocyte differentiation and inhibiting adipogenesis.

  16. Knockdown of NYGGF4 (PID1) rescues insulin resistance and mitochondrial dysfunction induced by FCCP in 3T3-L1 adipocytes.

    PubMed

    Shi, Chun-Mei; Wang, Yu-Mei; Zhang, Chun-Mei; Qiu, Jie; Shen, Ya-Hui; Zhu, Jin-Gai; Chen, Lin; Xu, Guang-Feng; Zhao, Ya-Ping; Ji, Chen-Bo; Guo, Xi-Rong

    2012-11-01

    NYGGF4 is a recently identified gene that is involved in obesity-associated insulin resistance. Previous data from this laboratory have demonstrated that NYGGF4 overexpression might contribute to the development of insulin resistance (IR) and to mitochondrial dysfunction. Additionally, NYGGF4 knockdown enhanced insulin sensitivity and mitochondrial function in 3T3-L1 adipocytes. We designed this study to determine whether silencing of NYGGF4 in 3T3-L1 adipocytes could rescue the effect of insulin sensitivity and mitochondrial function induced by the cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP), a mitochondrion uncoupler, to ascertain further the mechanism of NYGGF4 involvement in obesity-associated insulin resistance. We found that 3T3-L1 adipocytes, incubated with 5μM FCCP for 12h, had decreased levels of insulin-stimulated glucose uptake and had impaired insulin-stimulated GLUT4 translocation. Silencing also diminished insulin-stimulated tyrosinephosphorylation of IRS-1 and serine phosphorylation of Akt. This phenomenon contrasts with the effect of NYGGF4 knockdown on insulin sensitivity and describes the regulatory function of NYGGF4 in adipocytes insulin sensitivity. We next analyzed the mitochondrial function in NYGGF4-silenced adipocytes incubated with FCCP. NYGGF4 knockdown partly rescued the dissipation of mitochondrial mass, mitochondrial DNA, intracellular ATP synthesis, and intracellular reactive oxygen species (ROS) production occurred following the addition of FCCP, as well as inhibition of mitochondrial transmembrane potential (ΔΨm) in 3T3-L1 adipocytes incubated with FCCP. Collectively, our results suggested that addition of silencing NYGGF4 partly rescued the effect of insulin resistance and mitochondrial dysfunction in NYGGF4 silenced 3T3-L1 adipocytes incubated with FCCP, which might explain the involvement of NYGGF4-induced IR and the development of NYGGF4 in mitochondrial function.

  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. 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

  19. Chinese herbal medicine Yi-Gan-San decreases the lipid accumulation in mouse 3T3-L1 adipocytes by modulating the activities of transcription factors SREBP-1c and FoxO1.

    PubMed

    Izumi, Masayuki; Seki, Takashi; Iwasaki, Koh; Sakamoto, Kazuichi

    2009-09-01

    Abnormal lipid metabolism in adipose tissue is closely related to the occurrence and progression of a wide variety of metabolic syndromes. We have analyzed the pharmacological effects of Chinese herbal medicines on cell differentiation and lipid metabolism in adipocytes. Yi-Gan-San (YGS) is a Chinese herbal medicine that is effective in treating the behavioral and psychological symptoms of dementia; however, its physiological mechanism remains unclear. We analyzed the effects of YGS on lipid accumulation in mouse 3T3-L1 adipocytes. Adipocyte differentiation was induced in mouse 3T3-L1 preadipocytes by treatment with the mixture of dexamethasone, 3-iso-butyl-1-methylxanthine, and insulin, and cells were cultured for 8 days with Chinese herbal medicines, including YGS. YGS effectively reduced the lipid accumulation in the differentiated 3T3-L1 cells in a dose-dependent manner, but had no effect on cell viability. YGS also reduced the activity of glycerol-3-phosphate dehydrogenase, an enzyme involved in lipid synthesis. In contrast, YGS gave no noticeable effect on glucose uptake and fatty acid uptake in the differentiated 3T3-L1 cells. Moreover, we established the stably transfected 3T3-L1 cell lines, each of which expresses the luciferase reporter gene under the control of sterol regulatory element-binding protein-1c (SREBP-1c) or FoxO1. SREBP-1c is a transcription factor involved in fatty acid synthesis, and FoxO1 is a forkhead-type transcription factor involved in adipocyte differentiation. Using these cell lines, we showed that YGS reduced the transcriptional activity of SREBP-1c, whereas YGS increased the activity of FoxO1. Thus, YGS may suppress lipid synthesis and fat accumulation in adipocytes through modulating the activities of SREBP-1c and FoxO1.

  20. Effect of desacyl ghrelin, obestatin and related peptides on triglyceride storage, metabolism and GHSR signaling in 3T3-L1 adipocytes.

    PubMed

    Miegueu, Pierre; St Pierre, David; Broglio, Fabio; Cianflone, Katherine

    2011-02-01

    Acyl-ghrelin (AG), desacyl-ghrelin (DAG) and obestatin are all derived from the same gene transcript; however their plasma levels do not necessarily change in parallel. The influence of these peptides towards the development of obesity and their direct effects on adipocyte physiology has not been thoroughly investigated. This study was designed to evaluate the direct effects of peptides of the ghrelin family on preadipocyte proliferation, differentiation and adipocyte lipid and glucose metabolism in 3T3-L1 cells. 3T3 cells were treated with physiological peptide concentrations for 1 h to 9 days, and the relevant assays measured. In preadipocytes, AG, GHRP-6 and DAG stimulated proliferation, measured as (3)H-thymidine incorporation (up to 200%, P < 0.05), while all peptides stimulated differentiation (up to 300%, P < 0.01) as compared to standard differentiation conditions. In adipocytes, FA uptake was increased in a concentration-dependent manner especially with obestatin (three- to fourfold, P < 0.001) and DAG (three- to fivefold, P < 0.001). By contrast, glucose transport was unchanged. DAG and obestatin significantly decreased lipolysis measured as non-esterified fatty acid and glycerol release by 50%, P < 0.05-0.01 and 51%, P < 0.01, respectively. Interestingly, DAG stimulation of FA uptake was blocked with GHSR1 antagonist (D-lys(3))-GHRP-6 (P < 0.05), phospholipase C inhibitor U73122 and phosphatidylinositol-3-kinase inhibitor wortmannin (P < 0.001). Finally, in omental but not subcutaneous human adipose tissue, GHSR1 correlated with BMI (r = 0.549, P < 0.05) and insulin (r = 0.681, P < 0.01). Taken together, these results suggest that ghrelin-related peptides may directly affect adipose tissue metabolism.

  1. 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

  2. The Effect of Pericellular Oxygen Levels on Proteomic Profile and Lipogenesis in 3T3-L1 Differentiated Preadipocytes Cultured on Gas-Permeable Cultureware.

    PubMed

    Weiszenstein, Martin; Pavlikova, Nela; Elkalaf, Moustafa; Halada, Petr; Seda, Ondrej; Trnka, Jan; Kovar, Jan; Polak, Jan

    2016-01-01

    Pericellular oxygen concentration represents an important factor in the regulation of cell functions, including cell differentiation, growth and mitochondrial energy metabolism. Hypoxia in adipose tissue has been associated with altered adipokine secretion profile and suggested as a possible factor in the development of type 2 diabetes. In vitro experiments provide an indispensable tool in metabolic research, however, physical laws of gas diffusion make prolonged exposure of adherent cells to desired pericellular O2 concentrations questionable. The aim of this study was to investigate the direct effect of various O2 levels (1%, 4% and 20% O2) on the proteomic profile and triglyceride accumulation in 3T3-L1 differentiated preadipocytes using gas-permeable cultureware. Following differentiation of cells under desired pericellular O2 concentrations, cell lysates were subjected to two-dimensional gel electrophoresis and protein visualization using Coomassie blue staining. Spots showing differential expression under hypoxia were analyzed using matrix-assisted laser desorption/ionization mass spectrometry. All identified proteins were subjected to pathway analysis. We observed that protein expression of 26 spots was reproducibly affected by 4% and 1% O2 (17 upregulated and 9 downregulated). Pathway analysis showed that mitochondrial energy metabolism and triglyceride synthesis were significantly upregulated by hypoxia. In conclusion, this study demonstrated the direct effects of pericellular O2 levels on adipocyte energy metabolism and triglyceride synthesis, probably mediated through the reversed tricarboxylic acid cycle flux.

  3. The Effect of Pericellular Oxygen Levels on Proteomic Profile and Lipogenesis in 3T3-L1 Differentiated Preadipocytes Cultured on Gas-Permeable Cultureware

    PubMed Central

    Weiszenstein, Martin; Pavlikova, Nela; Elkalaf, Moustafa; Halada, Petr; Seda, Ondrej; Trnka, Jan; Kovar, Jan; Polak, Jan

    2016-01-01

    Pericellular oxygen concentration represents an important factor in the regulation of cell functions, including cell differentiation, growth and mitochondrial energy metabolism. Hypoxia in adipose tissue has been associated with altered adipokine secretion profile and suggested as a possible factor in the development of type 2 diabetes. In vitro experiments provide an indispensable tool in metabolic research, however, physical laws of gas diffusion make prolonged exposure of adherent cells to desired pericellular O2 concentrations questionable. The aim of this study was to investigate the direct effect of various O2 levels (1%, 4% and 20% O2) on the proteomic profile and triglyceride accumulation in 3T3-L1 differentiated preadipocytes using gas-permeable cultureware. Following differentiation of cells under desired pericellular O2 concentrations, cell lysates were subjected to two-dimensional gel electrophoresis and protein visualization using Coomassie blue staining. Spots showing differential expression under hypoxia were analyzed using matrix-assisted laser desorption/ionization mass spectrometry. All identified proteins were subjected to pathway analysis. We observed that protein expression of 26 spots was reproducibly affected by 4% and 1% O2 (17 upregulated and 9 downregulated). Pathway analysis showed that mitochondrial energy metabolism and triglyceride synthesis were significantly upregulated by hypoxia. In conclusion, this study demonstrated the direct effects of pericellular O2 levels on adipocyte energy metabolism and triglyceride synthesis, probably mediated through the reversed tricarboxylic acid cycle flux. PMID:27023342

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

    SciTech Connect

    Chung, Le Thi Kim; Hosaka, Toshio; Harada, Nagakatsu; Jambaldorj, Bayasgalan; Fukunaga, Keiko; Nishiwaki, Yuka; Teshigawara, Kiyoshi; Sakai, Tohru; Nakaya, Yutaka; Funaki, Makoto

    2010-01-01

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

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. Effect of long-term treatment of 3T3-L1 adipocytes with chlorate on the synthesis, glycosylation, intracellular transport and secretion of lipoprotein lipase.

    PubMed

    Masuno, H; Sakayama, K; Okuda, H

    1998-02-01

    Lipoprotein lipase (LPL) is synthesized and glycosylated in the endoplasmic reticulum (ER), transported through the Golgi to the cell surface, and finally secreted. To examine the role of heparan sulphate proteoglycans (HSPG) in the synthesis, activity, intracellular transport and secretion of LPL, 3T3-L1 adipocytes were cultured for 7 days in the presence of 20 mM chlorate, an inhibitor of sulphation of HSPG. Treatment of cells with 20 mM chlorate for 7 days caused a 55% decrease in LPL activity in the intracellular compartment and a 79% decrease in the cell-surface compartment. The synthetic rate of LPL in chlorate-treated cells was identical with that in control cells as determined by biosynthetic labelling. The study with endoglycosidase H (endo H) showed that the treatment with chlorate increased the proportion of LPL subunits which were totally endo H-sensitive. The study with a heparin-Sepharose column showed that 3T3-L1 adipocytes contained three forms of LPL. The first form, accounting for 35% of the LPL, did not bind to the heparin-Sepharose column and had little or no activity; the second form, accounting for 32%, bound to the column and was eluted with 0.4-0.75 M NaCl but had no activity; the third form, accounting for 33%, bound to the column and was eluted with 0.8-1.2 M NaCl and had activity. In chlorate-treated cells, the first form accounted for 66% of the LPL, the second form 15% and the third form 19%. When cells were incubated for 1 h with brefeldin A, which translocates Golgi proteins to the ER [J. Lippincott-Schwartz, L.C. Yuan, J.S. Banifacino and R.D. Klausner (1989) Cell 56, 801-813; J. Lippincott-Schwartz, J. Glickman, J.E. Donaldson, J. Robbins, T.E. Kreis, K.B. Seamon, M.P. Sheetz and R.D. Klausner (1991) J. Cell Biol. 112, 567-577], the chlorate-induced decrease in cellular LPL activity was restored. These findings indicate that LPL synthesized in chlorate-treated cells can be processed to be fully active, but chlorate-treated cells are

  12. Polychlorinated biphenyls (PCB 101, PCB 153 and PCB 180) alter leptin signaling and lipid metabolism in differentiated 3T3-L1 adipocytes

    SciTech Connect

    Ferrante, Maria C.; Amero, Paola; Santoro, Anna; Monnolo, Anna; Simeoli, Raffaele; Di Guida, Francesca; Mattace Raso, Giuseppina; Meli, Rosaria

    2014-09-15

    Non-dioxin-like polychlorinated biphenyls (NDL-PCBs) are highly lipophilic environmental contaminants that accumulate in lipid-rich tissues, such as adipose tissue. Here, we reported the effects induced by PCBs 101, 153 and 180, three of the six NDL-PCBs defined as indicators, on mature 3T3-L1 adipocytes. We observed an increase in lipid content, in leptin gene expression and a reduction of leptin receptor expression and signaling, when cells were exposed to PCBs, alone or in combination. These modifications were consistent with the occurrence of “leptin-resistance” in adipose tissue, a typical metabolic alteration related to obesity. Therefore, we investigated how PCBs affect the expression of pivotal proteins involved in the signaling of leptin receptor. We evaluated the PCB effect on the intracellular pathway JAK/STAT, determining the phosphorylation of STAT3, a downstream activator of the transcription of leptin gene targets, and the expression of SOCS3 and PTP1B, two important regulators of leptin resistance. In particular, PCBs 153 and 180 or all PCB combinations induced a significant reduction in pSTAT3/STAT3 ratio and an increase in PTP1B and SOCS3, evidencing an additive effect. The impairment of leptin signaling was associated with the reduction of AMPK/ACC pathway activation, leading to the increase in lipid content. These pollutants were also able to increase the transcription of inflammatory cytokines (IL-6 and TNFα). It is worthy to note that the PCB concentrations used are comparable to levels detectable in human adipose tissue. Our data strongly support the hypothesis that NDL-PCBs may interfere with the lipid metabolism contributing to the development of obesity and related diseases. - Highlights: • NDL-PCBs alter lipid content and metabolism in 3T3-L1 adipocytes. • Impairment of leptin signaling was induced by NDL-PCBs. • NDL-PCBs reduce AMPK and ACC activation. • NDL-PCBs induce the synthesis of pro-inflammatory cytokine by

  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. 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}.

  15. Aucubin, a naturally occurring iridoid glycoside inhibits TNF-α-induced inflammatory responses through suppression of NF-κB activation in 3T3-L1 adipocytes.

    PubMed

    Park, Kyoung Sik

    2013-06-01

    Obesity is closely associated with a state of chronic, low-grade inflammation characterized by abnormal cytokine production and activation of inflammatory signaling pathways in adipose tissue. Tumor necrosis factor (TNF)-α is chronically elevated in adipose tissues of obese rodents and humans. Increased levels of TNF-α are implicated in the induction of atherogenic adipokines, such as plasminogen activator inhibitor (PAI)-1, adipose-tissue-derived monocyte chemoattractant protein (MCP)-1, and interleukin (IL)-6. Aucubin, an iridoid glycoside existing in medicinal plants, has been reported to show an anti-inflammatory activity by suppression of TNF-α production in murine macrophages. The present study is aimed to investigate the effects of aucubin on TNF-α-induced atherogenic changes of the adipokines in differentiated 3T3-L1 cells. Aucubin significantly inhibited TNF-α-induced secretion and mRNA synthesis of the atherogenic adipokines including PAI-1, MCP-1, and IL-6. Further investigation of the molecular mechanism revealed that pretreatment with aucubin suppressed extracellular signal-regulated kinase (ERK) activation, inhibitory kappa Bα (IκBα) degradation, and subsequent nuclear factor kappa B (NF-κB) activation. These findings suggest that aucubin may improve obesity-induced atherosclerosis by attenuating TNF-α-induced inflammatory responses.

  16. 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.

  17. Production of angiotensin II receptors type one (AT1) and type two (AT2) during the differentiation of 3T3-L1 preadipocytes.

    PubMed

    Mallow, H; Trindl, A; Löffler, G

    2000-01-01

    During their development from progenitor cells, adipocytes not only express enzymatic activities necessary for the storage of triglycerides, but also achieve the capability to produce a number of endocrine factors such as leptin, tumor necrosis factor alpha (TNFalpha), complement factors, adiponectin/adipoQ, plasminogen activator inhibitor-1 (PAI-1), angiotensin II and others. Angiotensin II is produced from angiotensinogen by the proteolytic action of renin and angiotensin-converting enzyme; and several data point to the existence of a complete local renin-angiotensin system in adipose tissue, including angiotensin II receptors. In this study, we directly monitored the production of angiotensin II type one receptor (AT1) and angiotensin II type two receptor (AT2) proteins during the adipose conversion of murine 3T3-L1 preadipocytes by immunodetection with specific antibodies. AT1 receptors could be detected throughout the whole differentiation period. The strong AT2 signal in preadipocytes however was completely lost during the course of differentiation, which suggests that expression of AT2 receptors is inversely correlated to the adipose conversion program.

  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. 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.

  20. 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

  1. Arrest of endosome acidification by bafilomycin A1 mimics insulin action on GLUT4 translocation in 3T3-L1 adipocytes.

    PubMed Central

    Chinni, S R; Shisheva, A

    1999-01-01

    In insulin-sensitive fat and muscle cells, the major glucose transporter GLUT4 is constitutively sequestered in endosomal tubulovesicular membranes, and moves to the cell surface in response to insulin. While sequence information within GLUT4 appears to be responsible for its constitutive intracellular sequestration, the regulatory elements and mechanisms that enable this protein to achieve its unique sorting pattern under basal and insulin-stimulated conditions are poorly understood. We show here that arrest of endosome acidification in insulin-sensitive 3T3-L1 adipocytes by bafilomycin A1, a specific inhibitor of the vacuolar proton pump, results in the rapid and dose-dependent translocation of GLUT4 from the cell interior to the membrane surface; the effects of maximally stimulatory concentrations of bafilomycin A1 (400-800 nM) were equivalent to 50-65% of the effects of acute insulin treatment. Like insulin, bafilomycin A1 induced the redistribution of GLUT1 and Rab4, but not that of other proteins whose membrane localization has been shown to be insulin-insensitive. Studies to address the mechanism of this effect demonstrated that neither autophosphorylation nor internalization of the insulin receptor was altered by bafilomycin A1 treatment. Bafilomycin-induced GLUT4 translocation was not blocked by cell pretreatment with wortmannin. Taken together, these data indicate that arrest of endosome acidification mimics insulin action on GLUT4 and GLUT1 translocation by a mechanism distal to insulin receptor and phosphatidylinositol 3-kinase activation, and suggest an important role for endosomal pH in the membrane dynamics of the glucose transporters. PMID:10215598

  2. 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.

  3. 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.

  4. Chromium activates glucose transporter 4 trafficking and enhances insulin-stimulated glucose transport in 3T3-L1 adipocytes via a cholesterol-dependent mechanism.

    PubMed

    Chen, Guoli; Liu, Ping; Pattar, Guruprasad R; Tackett, Lixuan; Bhonagiri, Padma; Strawbridge, Andrew B; Elmendorf, Jeffrey S

    2006-04-01

    Evidence suggests that chromium supplementation may alleviate symptoms associated with diabetes, such as high blood glucose and lipid abnormalities, yet a molecular mechanism remains unclear. Here, we report that trivalent chromium in the chloride (CrCl3) or picolinate (CrPic) salt forms mobilize the glucose transporter, GLUT4, to the plasma membrane in 3T3-L1 adipocytes. Concomitant with an increase in GLUT4 at the plasma membrane, insulin-stimulated glucose transport was enhanced by chromium treatment. In contrast, the chromium-mobilized pool of transporters was not active in the absence of insulin. Microscopic analysis of an exofacially Myc-tagged enhanced green fluorescent protein-GLUT4 construct revealed that the chromium-induced accumulation of GLUT4-containing vesicles occurred adjacent to the inner cell surface membrane. With insulin these transporters physically incorporated into the plasma membrane. Regulation of GLUT4 translocation by chromium did not involve known insulin signaling proteins such as the insulin receptor, insulin receptor substrate-1, phosphatidylinositol 3-kinase, and Akt. Consistent with a reported effect of chromium on increasing membrane fluidity, we found that chromium treatment decreased plasma membrane cholesterol. Interestingly, cholesterol add-back to the plasma membrane prevented the beneficial effect of chromium on both GLUT4 mobilization and insulin-stimulated glucose transport. Furthermore, chromium action was absent in methyl-beta-cyclodextrin-pretreated cells already displaying reduced plasma membrane cholesterol and increased GLUT4 translocation. Together, these data reveal a novel mechanism by which chromium may enhance GLUT4 trafficking and insulin-stimulated glucose transport. Moreover, these findings at the level of the cell are consistent with in vivo observations of improved glucose tolerance and decreased circulating cholesterol levels after chromium supplementation.

  5. 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

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

    SciTech Connect

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

    2013-01-04

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

  7. 16K Fractionation of 3T3-L1 Adipocytes to Produce a Crude GLUT4-Containing Vesicle Fraction.

    PubMed

    Sadler, Jessica B A; Lamb, Christopher A; Gould, Gwyn W; Bryant, Nia J

    2016-03-01

    The insulin-sensitive pool of glucose transporter isoform 4 (GLUT4) can be isolated from total cell membranes using the 16K fractionation protocol, described here. This method produces a light membrane-containing supernatant that includes the insulin-sensitive pool of GLUT4 in GLUT4 storage vesicles. The 16K pellet fraction contains the heavy membranes (including the plasma membrane, mitochondria, nuclei, Golgi apparatus, and endoplasmic reticulum). The distribution of proteins between the two fractions is determined via immunoblotting. By subjecting insulin-stimulated versus unstimulated cells to this protocol, the mobilization of proteins out of the insulin-sensitive GLUT4 pool can be assessed.

  8. 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.

  9. 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.

  10. 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.

  11. Regulation of myosin IIA and filamentous actin during insulin-stimulated glucose uptake in 3T3-L1 adipocytes

    SciTech Connect

    Stall, Richard; Ramos, Joseph; Kent Fulcher, F.; Patel, Yashomati M.

    2014-03-10

    Insulin stimulated glucose uptake requires the colocalization of myosin IIA (MyoIIA) and the insulin-responsive glucose transporter 4 (GLUT4) at the plasma membrane for proper GLUT4 fusion. MyoIIA facilitates filamentous actin (F-actin) reorganization in various cell types. In adipocytes F-actin reorganization is required for insulin-stimulated glucose uptake. What is not known is whether MyoIIA interacts with F-actin to regulate insulin-induced GLUT4 fusion at the plasma membrane. To elucidate the relationship between MyoIIA and F-actin, we examined the colocalization of MyoIIA and F-actin at the plasma membrane upon insulin stimulation as well as the regulation of this interaction. Our findings demonstrated that MyoIIA and F-actin colocalized at the site of GLUT4 fusion with the plasma membrane upon insulin stimulation. Furthermore, inhibition of MyoII with blebbistatin impaired F-actin localization at the plasma membrane. Next we examined the regulatory role of calcium in MyoIIA-F-actin colocalization. Reduced calcium or calmodulin levels decreased colocalization of MyoIIA and F-actin at the plasma membrane. While calcium alone can translocate MyoIIA it did not stimulate F-actin accumulation at the plasma membrane. Taken together, we established that while MyoIIA activity is required for F-actin localization at the plasma membrane, it alone is insufficient to localize F-actin to the plasma membrane. - Highlights: • Insulin induces colocalization of MyoIIA and F-actin at the cortex in adipocytes. • MyoIIA is necessary but not sufficient to localize F-actin at the cell cortex. • MyoIIA-F-actin colocalization is regulated by calcium and calmodulin.

  12. 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

  13. 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.

  14. Forskolin Inhibits Lipopolysaccharide-Induced Modulation of MCP-1 and GPR120 in 3T3-L1 Adipocytes through an Inhibition of NFκB

    PubMed Central

    Chiadak, Jeanne Durendale; Arsenijevic, Tatjana; Verstrepen, Kevin; Gregoire, Françoise; Bolaky, Nargis; Delforge, Valérie; Flamand, Véronique

    2016-01-01

    In an obese state, Toll-like receptor-4 (TLR-4) upregulates proinflammatory adipokines secretion including monocyte chemotactic protein-1 (MCP-1) in adipose tissue. In contrast, G-protein coupled receptor 120 (GPR120) mediates antiobesity effects. The aim of this study was to determine the signaling pathway by which Forskolin (FK), a cyclic adenosine monophosphate- (cAMP-) promoting agent causing positive changes in body composition in overweight and obese adult men, affects MCP-1 and GPR120 expression during an inflammatory response induced by lipopolysaccharide (LPS) in adipocytes, such as in an obese state. 3T3-L1 cells differentiated into adipocytes (DC) were stimulated with LPS in the absence or presence of FK and inhibitors of TLR-4 and inhibitor of kappa B (IκBα). In DC, LPS increased MCP-1, TLR-4, and nuclear factor-κB1 (NFκB1) mRNA levels, whereas it decreased GPR120 mRNA levels. In DC, FK inhibited the LPS-induced increase in MCP-1, TLR-4, and NFκB1 mRNA levels and the LPS-induced decrease in GPR120 mRNA. BAY11-7082 and CLI-095 abolished these LPS-induced effects. In conclusion, FK inhibits LPS-induced increase in MCP-1 mRNA levels and decrease in GPR120 mRNA levels in adipocytes and may be a potential treatment for inflammation in obesity. Furthermore, TLR-4-induced activation of NFκB may be involved in the LPS-induced regulation of these genes. PMID:27881903

  15. Pulicaria jaubertii E. Gamal-Eldin reduces triacylglyceride content and modifies cellular antioxidant pathways in 3T3-L1 adipocytes.

    PubMed

    Al-Naqeb, Ghanya; Rousová, Jana; Kubátová, Alena; Picklo, Matthew J

    2016-06-25

    Levels of obesity in Middle Eastern countries are increasing. Phytochemicals have anti-obesogenic properties as evidenced by prevention of adipocyte differentiation and blocking triacylglyceride (TG) accumulation. In Yemen, Pulicaria jaubertii E. Gamal-Eldin (PJ) is a food additive and a traditional medicine. We tested the hypothesis that phytochemicals present in PJ inhibit adipocytic responses during differentiation of 3T3-L1 preadipocytes to adipocytes. Methanolic extracts of PJ did not block expression of fatty acid binding protein 4 (FABP4) a marker of differentiation but did inhibit TG accumulation. Treatment of 3T3-L1 preadipocytes increased NADPH:quinone oxidoreductase 1 (NQO1), a suppressor of TG accumulation. Further fractionation of the methanolic PJ extract with hexane and dichloromethane (DCM) demonstrated that bioactivity towards TG reduction and elevated expression of NQO1 and other antioxidant genes (glutamate cysteine ligase catalytic unit, glutathione disulfide reductase, glutathione peroxidase (GPx) 4 resided in the DCM fraction. Activity towards depleting GSH and elevating the expression of catalase and GPx3 were found in the DCM and hexane fractions. Analysis by gas chromatography and liquid chromatography coupled with mass spectrometry demonstrated the presence of catechin-like moieties in the DCM and methanolic fractions and suggest that these components were partially responsible for the bioactivity of these fractions. In summary, our data indicate that fractions derived PJ exhibit anti-adipogenic properties in part through the presence of catechin-like compounds.

  16. The release of soluble VAP-1/SSAO by 3T3-L1 adipocytes is stimulated by isoproterenol and low concentrations of TNFalpha.

    PubMed

    García-Vicente, S; Abella, A; Viguerie, N; Ros-Baró, A; Camps, M; Testar, X; Palacín, M; Zorzano, A; Marti, L

    2005-06-01

    Plasma level of the protein VAP-1/SSAO (Vascular Adhesion Protein-1/Semicarbazide-Sensitive Amine Oxidase) is increased in diabetes and/or obesity and may be related to vascular complications associated to these pathologies. The aim of this work was to complete a preceding study where we described the role played by some hormones or metabolites, implicated in diabetes and/or obesity, in the regulation of the release of VAP-1/SSAO by 3T3-L1 adipocytes. Here we focused on the previously observed effect produced by TNFalpha in the release of VAP-1/SSAO and studied the effect of a beta-adrenergic compound, isoproterenol. Both compounds stimulated the release of VAP-1/SSAO to the culture medium but had a different effect on the VAP-1/SSAO membrane form. While TNFalpha produced a decrease on VAP-1/SSAO membrane form content, isoproterenol did not modify it. We thus observed two different ways of regulation of the release of VAP-1/SSAO by 3T3-L1 adipocytes by metabolites implicated in diabetes and adipose tissue physiopathology. Our work permits a better understanding of this increased plasma VAP-1/SSAO levels observed in diabetes.

  17. Amelioration of Mitochondrial Dysfunction-Induced Insulin Resistance in Differentiated 3T3-L1 Adipocytes via Inhibition of NF-κB Pathways

    PubMed Central

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

    2014-01-01

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

  18. Buckwheat (Fagopyrum esculentum M.) sprout treated with methyl jasmonate (MeJA) improved anti-adipogenic activity associated with the oxidative stress system in 3T3-L1 adipocytes.

    PubMed

    Lee, Young-Jun; Kim, Kui-Jin; Park, Kee-Jai; Yoon, Bo-Ra; Lim, Jeong-Ho; Lee, Ok-Hwan

    2013-01-11

    Buckwheat sprouts contain various bioactive compounds including rutin which have a number of biological activities. We have previously shown that buckwheat sprouts (TBWE) treated with methyl jasmonate (MeJA) significantly increased the amount of phenolics and the antioxidant activity. The aim of this study was to demonstrate the effect of TBWE on anti-adipogenesis and pro-oxidant enzyme in 3T3-L1 adipocytes. We also evaluated the anti-oxidative activity of TBWE in adipocytes by using the nitroblue tetrazolium assay. Our data showed that TBWE markedly inhibited adipocyte differentiation and ROS production in 3T3-L1 cells compared with control groups. Moreover, TBWE has strongly shown the inhibition of adipogenic transcription factor as well as pro-oxidant enzymes. Together, we demonstrate that the MeJA treatment significantly increased the amount of phenolic compound, resulting in the suppression of adipogenesis and ROS production in the 3T3-L1 cells. These findings indicate that TBWE has the potential for anti-adipogenesis activity with anti-oxidative properties.

  19. 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.

  20. Recombinant C3adesArg/acylation stimulating protein (ASP) is highly bioactive: a critical evaluation of C5L2 binding and 3T3-L1 adipocyte activation.

    PubMed

    Cui, Wei; Lapointe, Marc; Gauvreau, Danny; Kalant, David; Cianflone, Katherine

    2009-10-01

    C5L2 is a recently identified receptor for C5a/C5adesArg, C3a and C3adesArg (ASP). C5a/C5adesArg bind with high affinity, with no identified activation. By contrast, some studies demonstrate C3a/ASP binding/activation to C5L2; others do not. Our aim is to critically evaluate ASP/C3adesArg-C5L2 binding and bioactivity. Cell-associated fluorescent-ASP (Fl-ASP) binding to C5L2 increased from transiently transfectedcells had similar results. Endogenous C5L2 expression increased from 3T3-L1 preadipocytes<3T3-L1 adipocytescells+/-Fl-ASP demonstrated background fluorescence only. In adherent C5L2-HEK (Fl-ASP sorted) and 3T3-L1 cells, blocking with 10% fetal calf serum, protamine sulfate or ovalbumin prevented (125)I-ASP non-specific binding (NSB, no cells), while albumin increased NSB. Binding to non-transfected HEK was comparable to NSB. Optimal specific binding was obtained at 20 degrees C (vs. 4 degrees C) in PBS or serum-free medium with K(d) 83.7+/-23.7 nM (C5L2-HEK), 66+/-15 nM (C5L2-CHO) and 76+/-14.3 nM (3T3-L1 preadipocytes); (125)I-C5a binding had greater affinity. Fl-ASP-C5L2 binding was comparable and concentration dependent (K(d) 31 nM (direct binding) and IC(50) 35 nM (competition binding) regardless of conditions). Recombinant ASP (rASP) produced in modified Escherichia coli Origami (DE3) (allowing folding and disulphide bridge formation), purified under non-denaturing conditions demonstrated 10x greater bioactivity vs. proteolytically derived plasma ASP for triglyceride synthesis and fatty acid uptake in 3T3-L1 adipocytes and preadipocytes while adipose tissue from C5L2 KO mice was non-responsive. rASP stimulation of adipocyte BODIPY-fatty acid uptake demonstrated EC(50) 115+/-93 nM and maximal stimulation of 413+/-33%, p<0.001. ASP binding has distinct characteristics that lead to C5L2 activation and increased

  1. Docosahexaenoic acid increases cellular adiponectin mRNA and secreted adiponectin protein, as well as PPARγ mRNA, in 3T3-L1 adipocytes.

    PubMed

    Oster, Richard T; Tishinsky, Justine M; Yuan, Zongfei; Robinson, Lindsay E

    2010-12-01

    Adiponectin, a protein secreted from adipose tissue, has been shown to have anti-diabetic and anti-inflammatory effects, but its regulation is not completely understood. Long-chain n-3 fatty acids eicosapentaenoic acid (20:5n-3; EPA) and docosahexaenoic acid (22:6n-3; DHA) may be involved in adiponectin regulation as they are potential ligands for peroxisome proliferator-activated receptor-γ (PPARγ), a key transcription factor for the adiponectin gene. To examine this, 3T3-L1 adipocytes were incubated with 125 µmol·L-1 EPA, DHA, palmitic, or oleic acids complexed to albumin, or with albumin alone (control) for 24 h. Adipocytes were also incubated for 24 h with EPA and DHA plus bisphenol-A-diglycidyl ether (BADGE), a PPARγ antagonist. Both EPA and DHA increased (p < 0.05) secreted adiponectin concentration compared with the control (44% and 102%, respectively), but did not affect cellular adiponectin protein content. Incubation with BADGE and DHA inhibited increases in secreted adiponectin protein, suggesting that DHA may act through a PPARγ-dependent mechanism. However, BADGE had no effect on EPA-induced increases in secreted adiponectin protein. Only DHA enhanced (p < 0.05) PPARγ and adiponectin mRNA expression compared wtih the control. Our results demonstrate that DHA increases cellular adiponectin mRNA and secreted adiponectin protein in 3T3-L1 adipocytes, possibly by a mechanism involving PPARγ. Moreover, DHA increased adiponectin concentration to a greater extent (40% more, p < 0.05) compared with EPA, emphasizing the need to consider the independent actions of EPA and DHA in adipocytes.

  2. 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.

  3. Effects of Varying Degrees of Intermittent Hypoxia on Proinflammatory Cytokines and Adipokines in Rats and 3T3-L1 Adipocytes

    PubMed Central

    Zhou, Qin; Zhu, Hui; Niu, Wen-yan; Feng, Jing; Wang, Yan; Cao, Jie; Chen, Bao-yuan

    2014-01-01

    Objectives Intermittent hypoxia (IH), resulted from recurring episodes of upper airway obstruction, is the hallmark feature and the most important pathophysiologic pathway of obstructive sleep apnea (OSA). IH is believed to be the most important factor causing systemic inflammation. Studies suggest that insulin resistance (IR) is positively associated with OSA. In this study, we hypothesized that the recurrence of IH might result in cellular and systemic inflammation, which was manifested through the levels of proinflammatory cytokines and adipokines after IH exposure, and because IR is linked with inflammation tightly, this inflammatory situation may implicate an IR status. Methods We developed an IH 3T3-L1 adipocyte and rat model respectively, recapitulating the nocturnal oxygen profile in OSA. In IH cells, nuclear factor kappa B (NF-κB) DNA binding reactions, hypoxia-inducible factor-1α (HIF-1α), glucose transporter-1 (Glut-1), necrosis factor alpha (TNF-α), interleukin (IL) -6, leptin, adiponectin mRNA transcriptional activities and protein expressions were measured. In IH rats, blood glucose, insulin, TNF-α, IL-6, leptin and adiponectin levels were analyzed. Results The insulin and blood glucose levels in rats and NF-κB DNA binding activities in cells had significantly statistical results described as severe IH>moderate IH>mild IH>sustained hypoxia>control. The mRNA and protein levels of HIF-1α and Glut-1 in severe IH group were the highest. In cellular and animal models, both the mRNA and protein levels of TNF-α, IL-6 and leptin were the highest in severe IH group, when the lowest in severe IH group for adiponectin. Conclusions Oxidative stress and the release of pro-inflammatory cytokines/adipokines, which are the systemic inflammatory markers, are associated with IH closely and are proportional to the severity of IH. Because IR and glucose intolerance are linked with inflammation tightly, our results may implicate the clinical relationships between

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

    SciTech Connect

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

    2008-02-01

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

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

    PubMed

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

    2014-05-01

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

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

    PubMed

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

    2014-06-01

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

  7. Stearoyl-CoA Desaturase 1 Is a Key Determinant of Membrane Lipid Composition in 3T3-L1 Adipocytes

    PubMed Central

    Hagen, Rachel; Vidal-Puig, Antonio

    2016-01-01

    Stearoyl-CoA desaturase 1 (SCD1) is a lipogenic enzyme important for the regulation of membrane lipid homeostasis; dysregulation likely contributes to obesity associated metabolic disturbances. SCD1 catalyses the Δ9 desaturation of 12-19 carbon saturated fatty acids to monounsaturated fatty acids. To understand its influence in cellular lipid composition we investigated the effect of genetic ablation of SCD1 in 3T3-L1 adipocytes on membrane microdomain lipid composition at the species-specific level. Using liquid chromatography/electrospray ionisation-tandem mass spectrometry, we quantified 70 species of ceramide, mono-, di- and trihexosylceramide, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, bis(monoacylglycero)phosphate, phosphatidylinositol and cholesterol in 3T3-L1 adipocytes in which a 90% reduction in scd1 mRNA expression was achieved with siRNA. Cholesterol content was unchanged although decreases in other lipids resulted in cholesterol accounting for a higher proportion of lipid in the membranes. This was associated with decreased membrane lateral diffusion. An increased ratio of 24:0 to 24:1 in ceramide, mono- and dihexosylceramide, and sphingomyelin likely also contributed to this decrease in lateral diffusion. Of particular interest, we observed a decrease in phospholipids containing arachidonic acid. Given the high degree of structural flexibility of this acyl chain this will influence membrane lateral diffusion, and is likely responsible for the transcriptional activation of Lands’ cycle enzymes lpcat3 and mboat7. Of relevance these profound changes in the lipidome were not accompanied by dramatic changes in gene expression in mature differentiated adipocytes, suggesting that adaptive homeostatic mechanisms to ensure partial maintenance of the biophysical properties of membranes likely occur at a post-transcriptional level. PMID:27632198

  8. 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.

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

    PubMed

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

    2015-07-01

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

  10. A link between adipogenesis and innate immunity: RNase-L promotes 3T3-L1 adipogenesis by destabilizing Pref-1 mRNA

    PubMed Central

    Wang, Yi-Ting; Chiang, Hou-Hsien; Huang, Ying-Shing; Hsu, Chia-Lang; Yang, Po-Jen; Juan, Hsueh-Fen; Yang, Wei-Shiung

    2016-01-01

    Ribonuclease L (RNase-L) is an endoribonuclease well known for its roles in innate immunity. Recently it has been shown to regulate several cellular functions by modulating the levels of specific mRNAs. In this study, we investigated whether RNase-L may regulate adipocyte functions. We showed that knockdown of RNase-L reduced 3T3-L1 adipocyte differentiation and lipid accumulation. After mRNA profiling, we found that upregulation of Pref-1 mRNA, an inhibitory regulator of adipogenesis, could explain the reduced adipocyte differentiation with RNase-L downregulation. The signaling molecules downstream to Pref-1, including focal adhesion kinase, extracellular signal-regulated kinases and SRY-box 9, were activated by RNase-L suppression. The presence of Pref-1 mRNA was detected in the mRNP complexes precipitated by anti-RNase-L antibody. Moreover, the Pref-1 mRNA decay rate was raised by elevated RNase-L ribonuclease activity. Finally, in stable cell clones with RNase-L silencing, suppression of Pref-1 mRNA by specific siRNA partially recovered the adipocyte differentiation phenotype. Consistent with our findings, meta-analysis of 45 public array datasets from seven independent studies showed a significant negative relationship between RNase-L and Pref-1 mRNA levels in mouse adipose tissues. Higher RNase-L and lower Pref-1 mRNAs were found in the adipose tissues of high-fat diet mice compared to those of ND mice. In line with this, our animal data also showed that the adipose tissues of obese rats contained higher RNase-L and lower Pref-1 expression in comparison to that of lean rats. This study demonstrated that Pref-1 mRNA is a novel substrate of RNase-L. RNase-L is involved in adipocyte differentiation through destabilizing Pref-1 mRNA, thus offering a new link among RNA metabolism, innate immunity and adipogenesis in obesity progression. PMID:27831565

  11. 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.

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

    PubMed Central

    1991-01-01

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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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

  1. Cucumis melo ssp. Agrestis var. Agrestis Ameliorates High Fat Diet Induced Dyslipidemia in Syrian Golden Hamsters and Inhibits Adipogenesis in 3T3-L1 Adipocytes

    PubMed Central

    Shankar, Kripa; Singh, Sumit K.; Kumar, Durgesh; Varshney, Salil; Gupta, Abhishek; Rajan, Sujith; Srivastava, Ankita; Beg, Muheeb; Srivastava, Anurag Kumar; Kanojiya, Sanjeev; Mishra, Dipak K.; Gaikwad, Anil N.

    2015-01-01

    Background: Cucumis melo ssp. agrestis var. agrestis (CMA) is a wild variety of C. melo. This study aimed to explore anti-dyslipidemic and anti-adipogenic potential of CMA. Materials and Methods: For initial anti-dyslipidemic and antihyperglycemic potential of CMA fruit extract (CMFE), male Syrian golden hamsters were fed a chow or high-fat diet with or without CMFE (100 mg/kg). Further, we did fractionation of this CMFE into two fractions namely; CMA water fraction (CMWF) and CMA hexane fraction (CMHF). Phytochemical screening was done with liquid chromatography-mass spectrometry LC- (MS)/MS and direct analysis in real time-MS to detect active compounds in the fractions. Further, high-fat diet fed dyslipidemic hamsters were treated with CMWF and CMHF at 50 mg/kg for 7 days. Results: Oral administration of CMFE and both fractions (CMWF and CMHF) reduced the total cholesterol, triglycerides, low‐density lipoprotein cholesterol, and very low‐density lipoprotein-cholesterol levels in high fat diet-fed dyslipidemic hamsters. CMHF also modulated expression of genes involved in lipogenesis, lipid metabolism, and reverse cholesterol transport. Standard biochemical diagnostic tests suggested that neither of fractions causes any toxicity to hamster liver or kidneys. CMFE and CMHF also decreased oil-red-O accumulation in 3T3-L1 adipocytes. Conclusion: Based on these results, it is concluded that CMA possesses anti-dyslipidemic and anti-hyperglycemic activity along with the anti-adipogenic activity. SUMMARY The oral administration of Cucumis melo agrestis fruit extract (CMFE) and its fractions (CMWF and CMHF) improved serum lipid profile in HFD fed dyslipidemic hamsters.CMFE, CMWF and CMHF significantly attenuated body weight gain and eWAT hypertrophy.The CMHF decreased lipogenesis in both liver and adipose tissue.CMFE and CMHF also inhibited adipogenesis in 3T3-L1 adipocytes. Abbreviation used: CMA: Cucumis melo ssp. agrestis var. agrestis, CMFE: CMA fruit extract, CMWF

  2. 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.

  3. 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.

  4. 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

  5. The Effect of Glucose Concentration and Sodium Phenylbutyrate Treatment on Mitochondrial Bioenergetics and ER Stress in 3T3-L1 Adipocytes

    PubMed Central

    Tanis, Ross M.; Piroli, Gerardo G.; Day, Stani D.; Frizzell, Norma

    2016-01-01

    While the 3T3-L1 adipocyte model is routinely used for the study of obesity and diabetes, the mitochondrial respiratory profile in normal versus high glucose has not been examined in detail. We matured adipocytes in normal (5 mM) or high (30 mM) glucose and insulin and examined the mitochondrial bioenergetics. We also assessed the requirement for the Unfolded Protein Response (UPR) and ER stress under these conditions. Basal respiration was ∼1.7-fold greater in adipocytes that had matured in 30 mM glucose; however, their ability to increase oxygen consumption in response to stress was impaired. Adipogenesis proceeded in both normal and high glucose with concomitant activation of the UPR, but only high glucose was associated with increased levels of ER stress and mitochondrial stress as observed by parallel increases in CHOP and protein succination. Treatment of adipocytes with sodium phenylbutyrate relieved mitochondrial stress through a reduction in mitochondrial respiration. Our data suggests that mitochondrial stress, protein succination and ER stress are uniquely linked in adipocytes matured in high glucose. PMID:25448036

  6. Purification and Characterization of Aporphine Alkaloids from Leaves of Nelumbo nucifera Gaertn and Their Effects on Glucose Consumption in 3T3-L1 Adipocytes

    PubMed Central

    Ma, Chengjun; Wang, Jinjun; Chu, Hongmei; Zhang, Xiaoxiao; Wang, Zhenhua; Wang, Honglun; Li, Gang

    2014-01-01

    Aporphine alkaloids from the leaves of Nelumbo nucifera Gaertn are substances of great interest because of their important pharmacological activities, particularly anti-diabetic, anti-obesity, anti-hyperlipidemic, anti-oxidant, and anti-HIV’s activities. In order to produce large amounts of pure alkaloid for research purposes, a novel method using high-speed counter-current chromatography (HSCCC) was developed. Without any initial cleanup steps, four main aporphine alkaloids, including 2-hydroxy-1-methoxyaporphine, pronuciferine, nuciferine and roemerine were successfully purified from the crude extract by HSCCC in one step. The separation was performed with a simple two-phase solvent system composed of n-hexane-ethyl acetate-methanol-acetonitrile-water (5:3:3:2.5:5, v/v/v/v/v). In each operation, 100 mg crude extracts was separated and yielded 6.3 mg of 2-hydroxy-1-methoxyaporphine (95.1% purity), 1.1 mg of pronuciferine (96.8% purity), 8.5 mg of nuciferine (98.9% purity), and 2.7 mg of roemerine (97.4%) respectively. The chemical structure of four aporphine alkaloids are identified by means of electrospray ionization MS (ESI-MS) and nuclear magnetic resonance (NMR) analysis. Moreover, the effects of four separated aporphine alkaloids on insulin-stimulated glucose consumption were examined in 3T3-L1 adipocytes. The results showed that 2-hydroxy-1-methoxyaporphine and pronuciferine increased the glucose consumption significantly as rosiglitazone did. PMID:24577311

  7. 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.

  8. 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

  9. Isomeric C12-alkamides from the roots of Echinacea purpurea improve basal and insulin-dependent glucose uptake in 3T3-L1 adipocytes.

    PubMed

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

    2014-12-01

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

  10. 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.

  11. 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.

  12. Mitochondrial dysfunction and activation of iNOS are responsible for the palmitate-induced decrease in adiponectin synthesis in 3T3L1 adipocytes

    PubMed Central

    Jeon, Min Jae; Leem, Jaechan; Ko, Myoung Seok; Jang, Jung Eun; Park, Hye-Sun; Kim, Hyun Sik; Kim, Mina; Kim, Eun Hee; Yoo, Hyun Ju; Lee, Chul-Ho; Park, In-Sun; Lee, Ki-Up

    2012-01-01

    Mitochondrial dysfunction and endoplasmic reticulum (ER) stress are considered the key determinants of insulin resistance. Impaired mitochondrial function in obese animals was shown to induce the ER stress response, resulting in reduced adiponectin synthesis in adipocytes. The expression of inducible nitric oxide synthase (iNOS) is increased in adipose tissues in genetic and dietary models of obesity. In this study, we examined whether activation of iNOS is responsible for palmitate-induced mitochondrial dysfunction, ER stress, and decreased adiponectin synthesis in 3T3L1 adipocytes. As expected, palmitate increased the expression levels of iNOS and ER stress response markers, and decreased mitochondrial contents. Treatment with iNOS inhibitor increased adiponectin synthesis and reversed the palmitate-induced ER stress response. However, the iNOS inhibitor did not affect the palmitate-induced decrease in mitochondrial contents. Chemicals that inhibit mitochondrial function increased iNOS expression and the ER stress response, whereas measures that increase mitochondrial biogenesis (rosiglitazone and adenoviral overexpression of nuclear respiratory factor-1) reversed them. Inhibition of mitochondrial biogenesis prevented the rosiglitazone-induced decrease in iNOS expression and increase in adiponectin synthesis. These results suggest that palmitate-induced mitochondrial dysfunction is the primary event that leads to iNOS induction, ER stress, and decreased adiponectin synthesis in cultured adipocytes. PMID:22809900

  13. 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

  14. Biogenesis, characterization, and the effect of vicenin-gold nanoparticles on glucose utilization in 3T3-L1 adipocytes: a bioinformatic approach to illuminate its interaction with PTP 1B and AMPK.

    PubMed

    Chockalingam, Shivashri; Thada, Rajarajeshwari; Dhandapani, Ramesh Kumar; Panchamoorthy, Rajasekar

    2015-01-01

    This study reported the synthesis of Vicenin-2 gold nanoparticles (VN-AuNPs) and evaluated their effect on the glucose utilization efficiency of 3T3-L1 adipocytes. The VN-AuNPs were characterized by microscopic, DLS and spectral analysis. The bio-reducing efficiency of Vicenin-2 (VN) was computed and confirmed by HPLC analysis. The stability of VN-AuNPs in various physiological media was explored. The cytotoxicity and glucose uptake assays were performed in 3T3-L1 adipocytes. The docking of VN with PTP1B and AMPK was also performed. The color change and UV absorption at 537 nm preliminarily confirmed the VN reduced gold nanoparticles. The VN-AuNPs appeared as spherical particles (57 nm) and face centered cubic crystals under TEM and XRD analysis, respectively. Its zeta potential was found to be -6.53 mV. The FT-IR spectra of VN and its AuNPs confirmed its stability. The computed reducing potential of VN was similar to the extent of VN utilized during the synthesis of VN-AuNPs. The VN-AuNPs showed a remarkable stability in different physiological media. At 100 µM concentration, VN-AuNPs displayed 78.21% cell viability. A concentration dependent increase in glucose uptake was noted in 3T3-L1 adipocytes when incubated with VN-AuNPs. The docking data revealed a strong interaction of VN with the binding pockets of PTP1B and AMPK. This demonstrates that the fabricated VN-AuNPs might enhance the intracellular VN availability mediated cellular glucose utilization and this would serve as a novel nanodrug for the management of diabetes.

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. Mogrol Derived from Siraitia grosvenorii Mogrosides Suppresses 3T3-L1 Adipocyte Differentiation by Reducing cAMP-Response Element-Binding Protein Phosphorylation and Increasing AMP-Activated Protein Kinase Phosphorylation

    PubMed Central

    Harada, Naoki; Ishihara, Mikako; Horiuchi, Hiroko; Ito, Yuta; Tabata, Hiromitsu; Suzuki, Yasushi A.; Nakano, Yoshihisa; Yamaji, Ryoichi; Inui, Hiroshi

    2016-01-01

    This study investigated the effects of mogrol, an aglycone of mogrosides from Siraitia grosvenorii, on adipogenesis in 3T3-L1 preadipocytes. Mogrol, but not mogrosides, suppressed triglyceride accumulation by affecting early (days 0–2) and late (days 4–8), but not middle (days 2–4), differentiation stages. At the late stage, mogrol increased AMP-activated protein kinase (AMPK) phosphorylation and reduced glycerol-3-phosphate dehydrogenase activity. At the early stage, mogrol promoted AMPK phosphorylation, inhibited the induction of CCAAT/enhancer-binding protein β (C/EBPβ; a master regulator of adipogenesis), and reduced 3T3-L1 cell contents (e.g., clonal expansion). In addition, mogrol, but not the AMPK activator AICAR, suppressed the phosphorylation and activity of the cAMP response element-binding protein (CREB), which regulates C/EBPβ expression. These results indicated that mogrol suppressed adipogenesis by reducing CREB activation in the initial stage of cell differentiation and by activating AMPK signaling in both the early and late stages of this process. PMID:27583359

  2. 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

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

    SciTech Connect

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

    1987-05-01

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

  4. Suppression of Very Early Stage Of Adipogenesis by Baicalein, a Plant-Derived Flavonoid through Reduced Akt-C/EBPα-GLUT4 Signaling-Mediated Glucose Uptake in 3T3-L1 Adipocytes

    PubMed Central

    2016-01-01

    Baicalein has been used as a Chinese medicine, and is an abundant plant flavonoid present in fruits and vegetables. Here, we examined the effects of baicalein in adipogenesis and investigated its molecular mechanism in adipocytes. Baicalein lowered the intracellular lipid accumulation and decreased the transcription levels of the adipocyte-specific genes in mouse 3T3-L1 adipocytes. Glucose uptake mediated by glucose transporter 4 (GLUT4) was reduced, causing down-regulation of the intracellular lipid accumulation. These reductions were also observed even when baicalein was added in only early stage of adipogenesis (0–2 days) of 6-day-adipogenesis. Chromatin immunoprecipitation assay showed that baicalein decreased the binding level of C/EBPα protein to the promoter region of the GLUT4 gene. Phosphorylation of Akt at 1 h after the initiation of adipogenesis was inhibited by the treatment with baicalein. Inhibition during only the first 1.5 h after the initiation of adipogenesis by baicalein or an Akt inhibitor was enough to decrease the lipid contents in the cells undergoing adipocyte differentiation for 6 days. These results indicate that baicalein decreased the intracellular lipid accumulation by down-regulation of glucose uptake via repression of Akt-C/EBPα-GLUT4 signaling in the very early stage of adipogenesis of 3T3-L1 adipocytes. PMID:27669565

  5. 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.

  6. 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.

  7. Five-parameter fluorescence imaging: wound healing of living Swiss 3T3 cells

    PubMed Central

    1987-01-01

    Cellular functions involve the temporal and spatial interplay of ions, metabolites, macromolecules, and organelles. To define the mechanisms responsible for completing cellular functions, we used methods that can yield both temporal and spatial information on multiple physiological parameters and chemical components in the same cell. We demonstrated that the combined use of selected fluorescent probes, fluorescence microscopy, and imaging methods can yield information on at least five separate cellular parameters and components in the same living cell. Furthermore, the temporal and spatial dynamics of each of the parameters and/or components can be correlated with one or more of the others. Five parameters were investigated by spectrally isolating defined regions of the ultraviolet, visible, and near-infrared spectrum based on five distinct fluorescent probes. The parameters included nuclei (Hoechst 33342), mitochondria (diIC1-[5] ), endosomes (lissamine rhodamine B-dextran), actin (fluorescein), and the cell volume Cy7- dextran). Nonmotile, confluent Swiss 3T3 cells did not show any detectable polarity of cell shape, or distribution of nuclei, endosomes, or mitochondria. These cells also organized a large percentage of the actin into stress fibers. In contrast, cells migrating into an in vitro wound exhibited at least two stages of reorganization of organelles and cytoplasm. During the first 3 h after wounding, the cells along the edge of the wound assumed a polarized shape, carried the nuclei in the rear of the cells, excluded endosomes and mitochondria from the lamellipodia, and lost most of the highly organized stress fibers. The cell showed a dramatic change between 3 and 7 h after producing the wound. The cells became highly elongated and motile; both the endosomes and the mitochondria penetrated into the lamellipodia, while the nuclei remained in the rear and the actin remained in less organized structures. Defining the temporal and spatial dynamics and

  8. 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.

  9. 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

  10. Nymphaea nouchali Burm. f. hydroalcoholic seed extract increases glucose consumption in 3T3-L1 adipocytes through activation of peroxisome proliferator-activated receptor gamma and insulin sensitization

    PubMed Central

    Parimala, Mabel; Debjani, M.; Vasanthi, Hannah Rachel; Shoba, Francis Gricilda

    2015-01-01

    Nymphaea nouchali Burm. f. (Family – Nymphaeaceae) is a well-known medicinal plant used in the Indian ayurvedic system of medicine for treating diabetes. The seeds especially have been prescribed for diabetes. The hydroalcoholic extract of N. nouchali seeds has been demonstrated to possess anti-hyperglycemic effects in diabetic rats, but the functional mechanism remains unknown. The nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARγ) is noted to play an important role in glucose and lipid homeostasis. This study was hence focused in evaluating the effect of the extract on PPARγ activation, adipocyte differentiation, and glucose consumption in 3T3-L1 cells. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), followed by adipogenesis assay using Oil Red O technique. Glucose consumption of preadipocytes and adipocytes in the presence of the extract was also determined. Real-time polymerase chain reaction was performed to identify the expression of genes involved in glucose consumption in the adipocytes. MTT assay confirmed the extract to be nontoxic, and Oil Red O staining confirmed enhanced adipocyte differentiation of 3T3-L1 cells in a dose-dependent manner. The extract also increased the expression of PPARγ target gene, which in turn enhanced the expression of GLUT-4. The data, therefore, suggests that N. nouchali seed extract promotes adipocyte differentiation and glucose consumption by inducing PPARγ activation, which in turn increases mRNA GLUT-4 expression and subsequently enhances insulin-responsiveness in insulin target tissues. PMID:26605160

  11. Nymphaea nouchali Burm. f. hydroalcoholic seed extract increases glucose consumption in 3T3-L1 adipocytes through activation of peroxisome proliferator-activated receptor gamma and insulin sensitization.

    PubMed

    Parimala, Mabel; Debjani, M; Vasanthi, Hannah Rachel; Shoba, Francis Gricilda

    2015-01-01

    Nymphaea nouchali Burm. f. (Family - Nymphaeaceae) is a well-known medicinal plant used in the Indian ayurvedic system of medicine for treating diabetes. The seeds especially have been prescribed for diabetes. The hydroalcoholic extract of N. nouchali seeds has been demonstrated to possess anti-hyperglycemic effects in diabetic rats, but the functional mechanism remains unknown. The nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARγ) is noted to play an important role in glucose and lipid homeostasis. This study was hence focused in evaluating the effect of the extract on PPARγ activation, adipocyte differentiation, and glucose consumption in 3T3-L1 cells. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), followed by adipogenesis assay using Oil Red O technique. Glucose consumption of preadipocytes and adipocytes in the presence of the extract was also determined. Real-time polymerase chain reaction was performed to identify the expression of genes involved in glucose consumption in the adipocytes. MTT assay confirmed the extract to be nontoxic, and Oil Red O staining confirmed enhanced adipocyte differentiation of 3T3-L1 cells in a dose-dependent manner. The extract also increased the expression of PPARγ target gene, which in turn enhanced the expression of GLUT-4. The data, therefore, suggests that N. nouchali seed extract promotes adipocyte differentiation and glucose consumption by inducing PPARγ activation, which in turn increases mRNA GLUT-4 expression and subsequently enhances insulin-responsiveness in insulin target tissues.

  12. Anti-Inflammatory Effect of Spirulina platensis in Macrophages Is Beneficial for Adipocyte Differentiation and Maturation by Inhibiting Nuclear Factor-κB Pathway in 3T3-L1 Adipocytes.

    PubMed

    Pham, Tho X; Lee, Ji-Young

    2016-06-01

    We previously showed that the organic extract of a blue-green alga, Spirulina platensis (SPE), had potent anti-inflammatory effects in macrophages. As the interplay between macrophages and adipocytes is critical for adipocyte functions, we investigated the contribution of the anti-inflammatory effects of SPE in macrophages to adipogenesis/lipogenesis in 3T3-L1 adipocytes. 3T3-L1 preadipocytes were treated with 10% conditioned medium from lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages (CMC) or LPS-stimulated, but SPE-pretreated, macrophages (CMS) at different stages of adipocyte differentiation. The expression of adipocyte differentiation markers, such as CCAAT/enhancer-binding protein α, peroxisome proliferator-activated receptor γ, and perilipin, was significantly repressed by CMC when added on day 3, while the repression was attenuated by CMS. Oil Red O staining confirmed that adipocyte maturation in CMS-treated cells, but not in CMC-treated cells, was equivalent to that of control cells. Nuclear translocation of nuclear factor κB (NF-κB) p65 was decreased by CMS compared to CMC. In lipid-laden adipocytes, CMC promoted the loss of lipid droplets, while CMS had minimal effects. Histone deacetylase 9 mRNA and protein levels were increased during adipocyte maturation, which were decreased by CMC. In conclusion, by cross-talking with adipocytes, the anti-inflammatory effects of SPE in macrophages promoted adipocyte differentiation/maturation, at least in part, by repressing the activation of NF-κB inflammatory pathways, which otherwise can be compromised in inflammatory conditions.

  13. Trans10,cis15 18:2 Isolated from Beef Fat Does Not Have the Same Anti-Adipogenic Properties as Trans10,cis12-18:2 in 3T3-L1 Adipocytes.

    PubMed

    Vahmani, Payam; Meadus, William J; Rolland, David C; Duff, Pascale; Dugan, Michael E R

    2016-11-01

    During ruminal biohydrogenation of α-linolenic acid, a non-conjugated non-methylene interrupted dienoic acid is formed containing a t10 double bond, namely t10,c15-18:2. The present study was designed to examine whether t10,c15-18:2 would exert similar anti-adipogenic effects compared to t10,c12-18:2 in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were treated with 35 or 70 µM of LNA, t10,c12-18:2, t10,c15-18:2, or bovine serum albumin (BSA) vehicle control for 120 h. Cellular triacylglycerol and protein were quantified using commercial colorimetric kits. Cells were analyzed for fatty acid composition and gene expression using gas chromatography and quantitative PCR, respectively. Trans10,cis12-18:2 decreased (P < 0.05) the adipocyte triacylglycerol (TAG) content, which was mainly related to a reduction in saturated fatty acids (SFA; e.g., 16:0 and 15:0) and cis monounsaturated fatty acids (c-MUFA; e.g., c9-16:1 and c9-18:1). Trans10,cis12 also decreased (P < 0.05) the expression of genes related to fatty acid synthesis (ACACA, FASN), delta-9 desaturation (SCD1), fatty acid elongation (ELOVL5), and fatty acid uptake (LPL) and upregulated (P < 0.05) the expression of the rate-liming enzyme involved in fatty acid β-oxidation (CPT1). In contrast, LNA and t10,c15-18:2 did not affect the gene expression and cellular content of the TAG, SFA, c-MUFA, or SCD1 indices in adipocytes. Our findings suggest that t10,c15-18:2, despite having structural similarity to t10,c12-18:2 (presence of a trans-10 double bond), does not exert anti-adipogenic effects in 3T3-L1 adipocytes.

  14. The Herbal Medicine KBH-1 Inhibits Fat Accumulation in 3T3-L1 Adipocytes and Reduces High Fat Diet-Induced Obesity through Regulation of the AMPK Pathway

    PubMed Central

    Lee, Ji-Hye; Kim, Taesoo; Lee, Jung-Jin; Lee, Kwang Jin; Kim, Hyun-Kyu; Yun, Bora; Jeon, Jongwook; Kim, Sang Kyum; Ma, Jin Yeul

    2015-01-01

    The aim of this study was to investigate whether a novel formulation of an herbal extract, KBH-1, has an inhibitory effect on obesity. To determine its anti-obesity effects and its underlying mechanism, we performed anti-obesity-related experiments in vitro and in vivo. 3T3-L1 preadipocytes were analyzed for lipid accumulation as well as the protein and gene expression of molecular targets involved in fatty acid synthesis. To determine whether KBH-1 oral administration results in a reduction in high-fat diet (HFD)-induced obesity, we examined five groups (n = 9) of C57BL/6 mice as follows: 10% kcal fat diet-fed mice (ND), 60% kcal fat diet-fed mice (HFD), HFD-fed mice treated with orlistat (tetrahydrolipstatin, marketed under the trade name Xenical), HFD-fed mice treated with 150 mg/kg KBH-1 (KBH-1 150) and HFD-fed mice treated with 300 mg/kg KBH-1 (KBH-1 300). During adipogenesis of 3T3-L1 cells in vitro, KBH-1 significantly reduced lipid accumulation and down-regulated the expression of master adipogenic transcription factors, including CCAAT/enhancer binding protein (C/EBP) β, C/EBP α and peroxisome proliferation-activity receptor (PPAR) γ, which led to the suppression of the expression of several adipocyte-specific genes and proteins. KBH-1 also markedly phosphorylated the adenosine monophosphate-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC). In addition, KBH-1-induced the inhibition effect on lipid accumulation and AMPK-mediated signal activation were decreased by blocking AMPK phosphorylation using AMPK siRNA. Furthermore, daily oral administration of KBH-1 resulted in dose-dependent decreases in body weight, fat pad mass and fat tissue size without systemic toxicity. These results suggest that KBH-1 inhibits lipid accumulation by down-regulating the major transcription factors of the adipogenesis pathway by regulating the AMPK pathway in 3T3-L1 adipocytes and in mice with HFD-induced obesity. These results implicate KBH-1, a safe herbal

  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. 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

  17. 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.

  18. 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.

  19. 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.

  20. Nickel-Refining Fumes Induced DNA Damage and Apoptosis of NIH/3T3 Cells via Oxidative Stress

    PubMed Central

    Wang, Yue; Wang, Sheng-Yuan; Jia, Li; Zhang, Lin; Ba, Jing-Chong; Han, Dan; Yu, Cui-Ping; Wu, Yong-Hui

    2016-01-01

    Although there have been numerous studies examining the toxicity and carcinogenicity of nickel compounds in humans and animals, its molecular mechanisms of action are not fully elucidated. In our research, NIH/3T3 cells were exposed to nickel-refining fumes at the concentrations of 0, 6.25, 12.50, 25, 50 and 100 μg/mL for 24 h. Cell viability, cell apoptosis, reactive oxygen species (ROS) level, lactate dehydrogenase (LDH) assay, the level of glutathione (GSH), activities of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) level were detected. The exposure of NIH/3T3 cells to nickel-refining fumes significantly reduced cell viability and induced cell apoptotic death in a dose-dependent manner. Nickel-refining fumes significantly increased ROS levels and induced DNA damage. Nickel-refining fumes may induce the changes in the state of ROS, which may eventually initiate oxidative stress, DNA damage and apoptosis of NIH/3T3 cells. PMID:27347984

  1. 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.

  2. 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.

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

    PubMed

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

    2012-12-01

    High fat diet induced insulin resistance correlates with dyslipidaemia and ectopic fat deposits in skeletal muscle and liver. The effects of Sutherlandia frutescens, an antidiabetic medicinal plant, on lipid metabolism were evaluated in an insulin resistant (IR) rat model and in 3 T3-preadipocytes. Wistar rats received normal diet (ND) or high fat diet (HFD). After the onset of IR in the HFD group, the rats were subdivided into two subgroups, which either continued with HFD or were treated with 50 mg S. frutescens/kg BW/day and HFD (HFD + SF). After 4 weeks, the HFD + SF rats had a significantly lower body weight than the HFD rats (p < 0.05). Blood plasma analysis showed a decrease in insulin, free fatty acids and triglycerides. Related changes in lipid parameters were observed in the liver, skeletal muscle and adipose tissue. To investigate the effects of S. frutescens on adipose tissue, 3 T3-L1 cells were used as a model. Treatment with S. frutescens led to a decrease in triglyceride accumulation, whilst glucose consumption and lactate production were increased (p < 0.05). These results indicate that S. frutescens directly affects mitochondrial activity and lipid biosynthesis in adipose tissue and provide a mechanism by which S. frutescens can restore insulin sensitivity by modulating fatty acid biosynthesis.

  4. 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.

  5. 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.

  6. 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

  7. Inhibitory effect of apocynin on methylglyoxal-mediated glycation in osteoblastic MC3T3-E1 cells.

    PubMed

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

    2015-04-01

    Methylglyoxal (MG), a highly reactive metabolite of hyperglycemia, can enhance protein glycation, oxidative stress or inflammation. The present study investigated the effects of apocynin on the mechanisms associated with MG toxicity in osteoblastic MC3T3-E1 cells. Pretreatment of MC3T3-E1 cells with apocynin prevented the MG-induced protein glycation and formation of intracellular reactive oxygen species and mitochondrial superoxide in MC3T3-E1 cells. In addition, apocynin increased glutathione levels and restored the activity of glyoxalase I inhibited by MG. These findings suggest that apocynin provide a protective action against MG-induced cell damage by reducing oxidative stress and by increasing the MG detoxification system. Apocynin treatment decreased the levels of proinflammatory cytokines such as tumor necrosis factor-α and interleukin-6 induced by MG. Additionally, the nitric oxide level reduced by MG was significantly increased by apocynin. These findings indicate that apocynin might exert its therapeutic effects via upregulation of glyoxalase system and antioxidant activity. Taken together, apocynin may prove to be an effective treatment for diabetic osteopathy.

  8. 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.

  9. Inhibitory Effects of Hwangryunhaedok-Tang in 3T3-L1 Adipogenesis by Regulation of Raf/MEK1/ERK1/2 Pathway and PDK1/Akt Phosphorylation

    PubMed Central

    Lee, Ji-Hye; Kim, Dong-Gun; Kim, Taesoo; Lee, Kwang Jin; Ma, Jin Yeul

    2013-01-01

    Hwangryunhaedok-tang (HRT) has been long used as traditional medicine in Asia. However, inhibitory role of HRT is unclear in early stage of 3T3-L1 adipocyte differentiation related to signaling. In the present study, we investigated the inhibitory effects of HRT on upstream signaling of peroxisome proliferation-activity receptor-γ (PPAR-γ) and CCAAT/enhancer binding protein-β (C/EBP-β) expression in differentiation of 3T3-L1 preadipocytes. We found that HRT significantly inhibited the adipocyte differentiation by downregulating several adipocyte-specific transcription factors including PPAR-γ, C/EBP-α, and C/EBP-β in 3T3-L1 preadipocytes. Furthermore, we observed that HRT markedly inhibited the differentiation media-mediated phosphorylation of Raf/extracellular mitogen-activated protein kinase 1 (MEK1)/signal-regulated protein kinase 1/2 (ERK1/2) and phosphorylation of phosphoinositide-dependent kinase 1 (PDK1)/Akt. These results indicate that anti-adipogenesis mechanism involves the downregulation of the major transcription factors of adipogenesis including PPAR-γ and C/EBP-α through inhibition of Raf/MEK1/ERK1/2 phosphorylation and PDK1/Akt phosphorylation by HRT. Furthermore, high performance liquid chromatography (HPLC) analysis showed HRT contains active antiobesity constituents such as palmatine, berberine, geniposide, baicalin, baicalein, and wogonin. Taken together, this study suggested that anti-adipogenesis effects of HRT were accounted by downregulation of Raf/MEK1/ERK1/2 pathway and PDK1/Akt pathway during 3T3-L1 adipocyte differentiation. PMID:23762131

  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. Two chalcones, 4-hydroxyderricin and xanthoangelol, stimulate GLUT4-dependent glucose uptake through the LKB1/AMP-activated protein kinase signaling pathway in 3T3-L1 adipocytes.

    PubMed

    Ohta, Mitsuhiro; Fujinami, Aya; Kobayashi, Norihiro; Amano, Akiko; Ishigami, Akihito; Tokuda, Harukuni; Suzuki, Nobutaka; Ito, Fumitake; Mori, Taisuke; Sawada, Morio; Iwasa, Koichi; Kitawaki, Jo; Ohnishi, Katsunori; Tsujikawa, Muneo; Obayashi, Hiroshi

    2015-07-01

    4-Hydroxyderricin (4HD) and xanthoangelol (XAG) are major components of n-hexane/ethyl acetate (5:1) extract of the yellow-colored stem juice of Angelica keiskei. 4-Hydroxyderricin and XAG have been reported to increase glucose transporter 4 (GLUT4)-dependent glucose uptake in 3T3-L1 adipocytes, but the detailed mechanism of this phenomenon remains unknown. This present study was aimed at clarifying the detailed mechanism by which 4HD and XAG increase GLUT4-dependent glucose uptake in 3T3-L1 adipocytes. Both 4HD and XAG increased glucose uptake and GLUT4 translocation to the plasma membrane. 4-Hydroxyderricin and XAG also stimulated the phosphorylation of 5' adenosine monophosphate-activated protein kinase (AMPK) and its downstream target acetyl-CoA carboxylase. In addition, phosphorylation of liver kinase B1 (LKB1), which acts upstream of AMPK, was also increased by 4HD and XAG treatment. Small interfering RNA knockdown of LKB1 attenuated 4HD- and XAG-stimulated AMPK phosphorylation and suppressed glucose uptake. These findings demonstrate that 4HD and XAG can increase GLUT4-dependent glucose uptake through the LKB1/AMPK signaling pathway in 3T3-L1 adipocytes.

  12. 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.

  13. 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.

  14. Carbonated apatites obtained by the hydrolysis of monetite: influence of carbonate content on adhesion and proliferation of MC3T3-E1 osteoblastic cells.

    PubMed

    Pieters, Ilse Y; Van den Vreken, Natasja M F; Declercq, Heidi A; Cornelissen, Maria J; Verbeeck, Ronald M H

    2010-04-01

    The influence of the carbonate content in apatites on the adhesion and the proliferation of MC3T3-E1 osteoblastic cells was investigated. B-type carbonated apatites (DCAps) were prepared by the hydrolysis of monetite (CaHPO(4), DCP) in solutions with a carbonate concentration ranging from 0.001 to 0.075 mol l(-1). Stoichiometric hydroxyapatite (DCAp0) was synthesized in carbonate-free solution. MC3T3-E1 cells were seeded on the compacted DCAps and cell adhesion and proliferation were analysed after 24h and 7 days, respectively, using a MTS assay and fluorescence microscopy. Cell adhesion tends to increase with increasing carbonate content for carbonate contents between 0 and 6.9 wt.% and levels out to an acceptable value (+ or - 50% compared to the control) for carbonate contents between 6.9 and 16.1 wt.%. Only DCAps with a carbonate content equal to or higher than 11% support high cell proliferation comparable to the control. On the latter DCAps, the cells have a spread morphology and form a near-confluent layer. A decrease in charge density and crystallinity at the apatite surface, as well as the formation of more spheroidal crystals with increasing carbonate content, might attribute to changes in composition and three-dimensional structure of the protein adsorption layer and hence to the observed cell behaviour. Consequently, only DCAps with a high carbonate content, mimicking early in vivo mineralization, are possible candidates for bone regeneration.

  15. 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.

  16. 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.

  17. Doses of Quercetin in the Range of Serum Concentrations Exert Delipidating Effects in 3T3-L1 Preadipocytes by Acting on Different Stages of Adipogenesis, but Not in Mature Adipocytes

    PubMed Central

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

    2015-01-01

    Scope. To determine whether doses of quercetin in the range of serum concentrations exert any effect on triacylglycerol accumulation in maturing preadipocytes and mature adipocytes. The influence on the expression of adipogenic markers as well as on gene expression and activity of enzymes involved in triacylglycerol metabolism were assessed. Methods and Results. 3T3-L1 preadipocytes were treated during differentiation and mature adipocytes for 24 hours with low doses (0.1–10 µM) of quercetin. Triacylglycerol content in both cell types and free fatty acid and glycerol in the incubation medium of mature adipocytes were measured spectrophotometrically. Gene and protein expression were assessed by RT-PCR and Western blot. LPL and FAS activities were quantified. During differentiation quercetin reduced triacylglycerol content at doses from 0.5 to 10 µM. 1 µM of quercetin reduced C/EBPβ gene expression, SREBP1 mature protein levels, and PPARγ gene expression. 10 µM of quercetin reduced LPL gene expression and PPARγ and SREBP1c expression. In mature adipocytes, only 10 µM of quercetin reduced triacylglycerol content. Lipogenic FAS expression and activity were reduced at this dose. Conclusion. Quercetin, in the range of serum concentrations, is able to inhibit adipogenesis, but higher doses, at least 10 µM, are needed to reduce fat accumulation in mature adipocytes. PMID:26180590

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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.

  3. Autophagy may protect MC3T3-E1 cells from fluoride-induced apoptosis.

    PubMed

    Wei, Min; Duan, Dongmei; Liu, Yujie; Wang, Zhigang; Li, Zhongli

    2014-06-01

    Fluoride is an essential trace element for all mammalian species; however, excess fluoride intake is known to be toxic to cells in animals and humans. The toxicity of fluoride is mainly exerted via induction of apoptosis. Autophagy is induced by numerous cytotoxic stimuli; however, it is often unclear whether, under specific conditions, autophagy has a pro‑survival or a pro‑apoptotic role. To answer this critical question, the present study assessed autophagy and apoptosis simultaneously in single cells. It was demonstrated that fluoride was able to inhibit cell proliferation and induce apoptosis and autophagy, whereas autophagy appeared to be protective. Further analysis revealed that MAPK/JNK‑dependent autophagy may be protective in fluoride‑induced apoptosis. It is anticipated that the presented single‑cell approach may be a powerful tool for gaining a quantitative understanding of the complex regulation of autophagy, its effect on cell fate and its association with other cellular pathways.

  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. 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.

  6. 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

  7. 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.

  8. 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.

  9. Insulin-IGF signaling affects cell transformation in the BALB/c 3T3 cell model

    PubMed Central

    Poburski, Doerte; Leovsky, Christiane; Boerner, Josefine Barbara; Szimmtenings, Luisa; Ristow, Michael; Glei, Michael; Thierbach, René

    2016-01-01

    The increased cancer mortality of diabetes type 2 patients is most likely an evidence of the tight connection between tumor development and energy metabolism. A major focus of today’s research is still the identification of key proteins of both diseases and the development of corresponding inhibitors. In this study we combined the two-stage BALB/c-3T3 cell transformation assay (BALB-CTA) with the IR/IGF-1R inhibitor OSI-906 (linsitinib) and analyzed alterations in protein activity and energy parameters in non-transformed as well as transformed cells. OSI-906 successfully inhibited the phosphorylation of IR/IGF-1R and decreased cell growth in non-transformed cells. In the BALB-CTA, a permanent treatment with OSI-906 reduced cellular transformation dose-dependently, whereas a temporary treatment gave evidence for a preventive effect in the promotion phase. Furthermore, even though several key proteins were affected, it was possible to show that the phosphorylation of GSK3, Erk 1/2 and the S6 protein are not crucial for the cell foci reducing effect of OSI-906. Taken together, the BALB-CTA confirmed results of OSI-906 from animal studies and enhanced the knowledge of its mode of action. Therefore, the BALB-CTA offers the opportunity to analyze alterations in the transformation process more precisely and will be helpful to identify effective cancer treatments. PMID:27849005

  10. 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.

  11. Ricin Toxicity in BALB/C 3T3 Cells: Peptide Biomarkers of Exposure

    DTIC Science & Technology

    2011-06-01

    formic acid ). The column was connected to a Finnigan LTQ tandem ion trap MS fitted with a nanospray ESI source operated at 1.82 kV with a collision...a humidified atmosphere of 5% CO2 in air for 24 h prior to treatment. Exposures were performed 24 ± 2 h after seeding the 75 cm2 flasks. Three...were incubated at 37 °C in a humidified atmosphere of 5% C02 in air for another 48 h. Cell harvesting was carried out 48 h ± 0.5 h post-exposure

  12. Modification of heterotrimeric G-proteins in Swiss 3T3 cells stimulated with Pasteurella multocida toxin.

    PubMed

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

    2012-01-01

    Many bacterial toxins covalently modify components of eukaryotic signalling pathways in a highly specific manner, and can be used as powerful tools to decipher the function of their molecular target(s). The Pasteurella multocida toxin (PMT) mediates its cellular effects through the activation of members of three of the four heterotrimeric G-protein families, G(q), G(12) and G(i). PMT has been shown by others to lead to the deamidation of recombinant Gα(i) at Gln-205 to inhibit its intrinsic GTPase activity. We have investigated modification of native Gα subunits mediated by PMT in Swiss 3T3 cells using 2-D gel electrophoresis and antibody detection. An acidic change in the isoelectric point was observed for the Gα subunit of the G(q) and G(i) families following PMT treatment of Swiss 3T3 cells, which is consistent with the deamidation of these Gα subunits. Surprisingly, PMT also induced a similar modification of Gα(11), a member of the G(q) family of G-proteins that is not activated by PMT. Furthermore, an alkaline change in the isoelectric point of Gα(13) was observed following PMT treatment of cells, suggesting differential modification of this Gα subunit by PMT. G(s) was not affected by PMT treatment. Prolonged treatment with PMT led to a reduction in membrane-associated Gα(i), but not Gα(q). We also show that PMT inhibits the GTPase activity of G(q).

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

    PubMed

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

    2012-03-01

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

  14. MicroRNA-15a fine-tunes the level of Delta-like 1 homolog (DLK1) in proliferating 3T3-L1 preadipocytes

    SciTech Connect

    Andersen, Ditte C.; Schneider, Mikael; Eskildsen, Tilde; Teisner, Borge; and others

    2010-06-10

    Delta like 1 homolog (Dlk1) exists in both transmembrane and soluble molecular forms, and is implicated in cellular growth and plays multiple roles in development, tissue regeneration, and cancer. Thus, DLK1 levels are critical for cell function, and abnormal DLK1 expression can be lethal; however, little is known about the underlying mechanisms. We here report that miR-15a modulates DLK1 levels in preadipocytes thus providing a mechanism for DLK1 regulation that further links it to cell cycle arrest and cancer since miR-15a is deregulated in these processes. In preadipocytes, miR-15a increases with cell density, and peaks at the same stage where membrane DLK1{sup M} and soluble DLK1{sup S} are found at maximum levels. Remarkably, miR-15a represses the amount of all Dlk1 variants at the mRNA level but also the level of DLK1{sup M} protein while it increases the amount of DLK1{sup S} supporting a direct repression of DLK1 and a parallel effect on the protease that cleaves off the DLK1 from the membrane. In agreement with previous studies, we found that miR-15a represses cell numbers, but additionally, we report that miR-15a also increases cell size. Conversely, anti-miR-15a treatment decreases cell size while increasing cell numbers, scenarios that were completely rescued by addition of purified DLK1{sup S}. Our data thus imply that miR-15a regulates cell size and proliferation by fine-tuning Dlk1 among others, and further emphasize miR-15a and DLK1 levels to play important roles in growth signaling networks.

  15. Decreased fat accumulation in 3T3-L1 pre-adipocytes treated with extracts of heat processed soy flour and breads

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The antiadipogenic effects of a gluten-free soy bread developed from soy flour pretreated by germination (GS), steaming (SS), or roasting (RS) were evaluated in an in vitro adipocyte cell model. Previously we had shown that soy bread prepared by GS, SS or RS flours had acceptable sensory attributes ...

  16. 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