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Sample records for t3 receptor induces

  1. Overexpression of the Mitochondrial T3 Receptor Induces Skeletal Muscle Atrophy during Aging

    PubMed Central

    Casas, François; Pessemesse, Laurence; Grandemange, Stéphanie; Seyer, Pascal; Baris, Olivier; Gueguen, Naïg; Ramonatxo, Christelle; Perrin, Florence; Fouret, Gilles; Lepourry, Laurence; Cabello, Gérard; Wrutniak-Cabello, Chantal

    2009-01-01

    In previous studies, we characterized a new hormonal pathway involving a mitochondrial T3 receptor (p43) acting as a mitochondrial transcription factor. In in vitro and in vivo studies, we have shown that p43 increases mitochondrial transcription and mitochondrial biogenesis. In addition, p43 overexpression in skeletal muscle stimulates mitochondrial respiration and induces a shift in metabolic and contractile features of muscle fibers which became more oxidative. Here we have studied the influence of p43 overexpression in skeletal muscle of mice during aging. We report that p43 overexpression initially increased mitochondrial mass. However, after the early rise in mitochondrial DNA occurring at 2 months of age in transgenic mice, we observed a progressive decrease of mitochondrial DNA content which became 2-fold lower at 23 months of age relatively to control animals. Moreover, p43 overexpression induced an oxidative stress characterized by a strong increase of lipid peroxidation and protein oxidation in quadriceps muscle, although antioxidant enzyme activities (catalase and superoxide dismutase) were stimulated. In addition, muscle atrophy became detectable at 6 months of age, probably through a stimulation of the ubiquitin proteasome pathway via two muscle-specific ubiquitin ligases E3, Atrogin-1/MAFbx and MuRF1. Taken together, these results demonstrate that a prolonged stimulation of mitochondrial activity induces muscle atrophy. In addition, these data underline the importance of a tight control of p43 expression and suggest that a deregulation of the direct T3 mitochondrial pathway could be one of the parameters involved in the occurrence of sarcopenia. PMID:19462004

  2. Overexpression of the Mitochondrial T3 Receptor p43 Induces a Shift in Skeletal Muscle Fiber Types

    PubMed Central

    Casas, François; Pessemesse, Laurence; Grandemange, Stéphanie; Seyer, Pascal; Gueguen, Naïg; Baris, Olivier; Lepourry, Laurence; Cabello, Gérard; Wrutniak-Cabello, Chantal

    2008-01-01

    In previous studies, we have characterized a new hormonal pathway involving a mitochondrial T3 receptor (p43) acting as a mitochondrial transcription factor and consequently stimulating mitochondrial activity and mitochondrial biogenesis. We have established the involvement of this T3 pathway in the regulation of in vitro myoblast differentiation.We have generated mice overexpressing p43 under control of the human α-skeletal actin promoter. In agreement with the previous characterization of this promoter, northern-blot and western-blot experiments confirmed that after birth p43 was specifically overexpressed in skeletal muscle. As expected from in vitro studies, in 2-month old mice, p43 overexpression increased mitochondrial genes expression and mitochondrial biogenesis as attested by the increase of mitochondrial mass and mt-DNA copy number. In addition, transgenic mice had a body temperature 0.8°C higher than control ones and displayed lower plasma triiodothyronine levels. Skeletal muscles of transgenic mice were redder than wild-type animals suggesting an increased oxidative metabolism. In line with this observation, in gastrocnemius, we recorded a strong increase in cytochrome oxidase activity and in mitochondrial respiration. Moreover, we observed that p43 drives the formation of oxidative fibers: in soleus muscle, where MyHC IIa fibers were partly replaced by type I fibers; in gastrocnemius muscle, we found an increase in MyHC IIa and IIx expression associated with a reduction in the number of glycolytic fibers type IIb. In addition, we found that PGC-1α and PPARδ, two major regulators of muscle phenotype were up regulated in p43 transgenic mice suggesting that these proteins could be downstream targets of mitochondrial activity. These data indicate that the direct mitochondrial T3 pathway is deeply involved in the acquisition of contractile and metabolic features of muscle fibers in particular by regulating PGC-1α and PPARδ. PMID:18575627

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

    SciTech Connect

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

    2009-06-01

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

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

    SciTech Connect

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

    1986-05-29

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

  5. Thyroid Hormone T3 Counteracts STZ Induced Diabetes in Mouse

    PubMed Central

    Madaro, Luca; Ranieri, Danilo; Lupoi, Lorenzo; Stigliano, Antonio; Torrisi, Maria Rosaria; Bouchè, Marina; Toscano, Vincenzo; Misiti, Silvia

    2011-01-01

    This study intended to demonstrate that the thyroid hormone T3 counteracts the onset of a Streptozotocin (STZ) induced diabetes in wild type mice. To test our hypothesis diabetes has been induced in Balb/c male mice by multiple low dose Streptozotocin injection; and a group of mice was contemporaneously injected with T3. After 48 h mice were tested for glucose tolerance test, insulin serum levels and then sacrified. Whole pancreata were utilized for morphological and biochemical analyses, while protein extracts and RNA were utilized for expression analyses of specific molecules. The results showed that islets from T3 treated mice were comparable to age- and sex-matched control, untreated mice in number, shape, dimension, consistency, ultrastructure, insulin and glucagon levels, Tunel positivity and caspases activation, while all the cited parameters and molecules were altered by STZ alone. The T3-induced pro survival effect was associated with a strong increase in phosphorylated Akt. Moreover, T3 administration prevented the STZ-dependent alterations in glucose blood level, both during fasting and after glucose challenge, as well as in insulin serum level. In conclusion we demonstrated that T3 could act as a protective factor against STZ induced diabetes. PMID:21637761

  6. Analysis of the functional state of T3 nuclear receptors expressed in thyroid cells.

    PubMed

    Selmi-Ruby, S; Rousset, B

    1996-05-17

    T3 nuclear receptors (TR) are present in thyroid cells. We have analyzed the ability of thyroid TR to function as transcriptional regulators. Studies were performed on pig thyrocytes in primary culture. Messenger RNA corresponding to TR alpha 1, alpha 2 and beta were detected in pig thyrocytes by RT-PCR and Northern blot; the alpha 2 mRNA was more abundant than the alpha 1 mRNA. Thyrocytes were transiently transfected with different plasmids containing the CAT (chloramphenicol acetyl transferase) gene placed under the control of different promoters (delta MTV, TK or delta SV40) and bearing a thyroid hormone response element, TREp or TRE DR + 4. It was found that TSH induced a concentration-dependent increase of the transfection efficiency, an effect reproduced by (Bu)2cAMP and Forskolin. Cells transfected with either delta MTV-, TK- or delta SV40-TREp-CAT expressed similar basal CAT activities. Addition of T3 produced a 3-fold increase of CAT activity expressed from each of these vectors. In contrast, CAT activity expressed from a vector containing the TRE DR + 4 was decreased by about 50% by T3. Thus, TREp and TRE DR + 4 gave distinct responses. These data demonstrate that TR physiologically expressed in thyroid cells can act as transcriptional regulators in a T3-dependent manner. This finding directly substantiates the concept of autocrine regulatory actions of thyroid hormones. PMID:8793858

  7. Interleukin 1 amplifies receptor-mediated activation of phospholipase A2 in 3T3 fibroblasts.

    PubMed Central

    Burch, R M; Connor, J R; Axelrod, J

    1988-01-01

    Human recombinant interleukin 1 alpha (IL-1 alpha) and IL-1 beta stimulated prostaglandin E2 synthesis in 3T3 fibroblasts in a time- and concentration-dependent manner. Enhanced prostaglandin E2 synthesis after IL-1 treatment was apparent by 1 hr and continued to increase for at least 2 days. Half-maximal stimulation occurred at 0.5 pM IL-1 alpha or IL-1 beta, and both interleukins were equally effective, with maximal stimulation occurring in response to 5-10 pM IL-1. In contrast to IL-1, bradykinin stimulation of prostaglandin E2 synthesis is rapid; its effect is maximal by 5 min. In cells that had been pretreated with IL-1 for 24 hr, prostaglandin E2 synthesis in response to bradykinin was amplified more than 10-fold. IL-1 also amplified the receptor-mediated formation of prostaglandin E2 by bombesin and thrombin. The lymphokine did not affect bradykinin receptor number or affinity. IL-1 treatment induced phospholipase A2 and cyclooxygenase but not phospholipase C or prostaglandin E isomerase. It also enhanced bradykinin-stimulated GTPase activity, suggesting possible induction of the GTP-binding regulatory protein coupled to the bradykinin receptor. Thus, IL-1 enhanced receptor-mediated release of prostaglandin E2 in response to bradykinin, bombesin, and thrombin by increasing the cellular levels of phospholipase A2, cyclooxygenase, and GTP-binding regulatory protein(s). PMID:2901097

  8. High-affinity receptors for peptides of the bombesin family in Swiss 3T3 cells

    SciTech Connect

    Zachary, I.; Rozengurt, E.

    1985-11-01

    Gastrin-releasing peptide (GRP) labeled with /sup 125/I at tyrosine-15 (/sup 125/I-GRP) binds to intact quiescent Swiss 3T3 cells in a specific and saturable manner. Scatchard analysis indicates the presence of a single class of high-affinity binding sites of Kd = 0.5 X 10(-9) M and a value for the number of sites per cell of about 100,000. /sup 125/I-GRP binding was not inhibited by other mitogens for these cells, and cell lines that are mitogenically unresponsive to GRP do not exhibit specific GRP binding. Structure-activity relationships show a close parallel between the ability of a range of GRP-related peptides to both inhibit GRP binding and to stimulate mitogenesis. Further, GRP binding is selectively blocked in a competitive fashion by a novel bombesin antagonist, (D-Arg1, D-Pro2, D-Trp7,9, Leu11) substance P. In addition, this compound selectively inhibits GRP and bombesin-induced mitogenesis. These results demonstrate that the mitogenic response of Swiss 3T3 cells to peptides of the bombesin family is mediated by a class of receptors distinct from those of other mitogens for these cells.

  9. 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}.

  10. 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.

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

    PubMed

    Masiello, Lisa M; Fotos, Joseph S; Galileo, Deni S; Karin, Norman 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. PMID:16487757

  12. 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.

  13. T3-induced liver AMP-activated protein kinase signaling: Redox dependency and upregulation of downstream targets

    PubMed Central

    Videla, Luis A; Fernández, Virginia; Cornejo, Pamela; Vargas, Romina; Morales, Paula; Ceballo, Juan; Fischer, Alvaro; Escudero, Nicolás; Escobar, Oscar

    2014-01-01

    AIM: To investigate the redox dependency and promotion of downstream targets in thyroid hormone (T3)-induced AMP-activated protein kinase (AMPK) signaling as cellular energy sensor to limit metabolic stresses in the liver. METHODS: Fed male Sprague-Dawley rats were given a single ip dose of 0.1 mg T3/kg or T3 vehicle (NaOH 0.1 N; controls) and studied at 8 or 24 h after treatment. Separate groups of animals received 500 mg N-acetylcysteine (NAC)/kg or saline ip 30 min prior T3. Measurements included plasma and liver 8-isoprostane and serum β-hydroxybutyrate levels (ELISA), hepatic levels of mRNAs (qPCR), proteins (Western blot), and phosphorylated AMPK (ELISA). RESULTS: T3 upregulates AMPK signaling, including the upstream kinases Ca2+-calmodulin-dependent protein kinase kinase-β and transforming growth factor-β-activated kinase-1, with T3-induced reactive oxygen species having a causal role due to its suppression by pretreatment with the antioxidant NAC. Accordingly, AMPK targets acetyl-CoA carboxylase and cyclic AMP response element binding protein are phosphorylated, with the concomitant carnitine palmitoyltransferase-1α (CPT-1α) activation and higher expression of peroxisome proliferator-activated receptor-γ co-activator-1α and that of the fatty acid oxidation (FAO)-related enzymes CPT-1α, acyl-CoA oxidase 1, and acyl-CoA thioesterase 2. Under these conditions, T3 induced a significant increase in the serum levels of β-hydroxybutyrate, a surrogate marker for hepatic FAO. CONCLUSION: T3 administration activates liver AMPK signaling in a redox-dependent manner, leading to FAO enhancement as evidenced by the consequent ketogenic response, which may constitute a key molecular mechanism regulating energy dynamics to support T3 preconditioning against ischemia-reperfusion injury. PMID:25516653

  14. TNF-α Induces Caspase-1 Activation Independently of Simultaneously Induced NLRP3 in 3T3-L1 Cells.

    PubMed

    Furuoka, Mana; Ozaki, Kei-Ichi; Sadatomi, Daichi; Mamiya, Sayaka; Yonezawa, Tomo; Tanimura, Susumu; Takeda, Kohsuke

    2016-12-01

    The intracellular cysteine protease caspase-1 is critically involved in obesity-induced inflammation in adipose tissue. A substantial body of evidence from immune cells, such as macrophages, has shown that caspase-1 activation depends largely on a protein complex, called the NLRP3 inflammasome, which consists of the NOD-like receptor (NLR) family protein NLRP3, the adaptor protein ASC, and caspase-1 itself. However, it is not fully understood how caspase-1 activation is regulated within adipocytes upon inflammatory stimuli. In this study, we show that TNF-α-induced activation of caspase-1 is accompanied by robust induction of NLRP3 in 3T3-L1 adipocytes but that caspase-1 activation may not depend on the NLRP3 inflammasome. Treatment of 3T3-L1 cells with TNF-α induced mRNA expression and activation of caspase-1. Although the basal expression of NLRP3 and ASC was undetectable in unstimulated cells, TNF-α strongly induced NLRP3 expression but did not induce ASC expression. Interestingly, inhibitors of the ERK MAP kinase pathway strongly suppressed NLRP3 expression but did not suppress the expression and activation of caspase-1 induced by TNF-α, suggesting that NLRP3 is dispensable for TNF-α-induced caspase-1 activation. Moreover, we did not detect the basal and TNF-α-induced expression of other NLR proteins (NLRP1a, NLRP1b, and NLRC4), which do not necessarily require ASC for caspase-1 activation. These results suggest that TNF-α induces caspase-1 activation in an inflammasome-independent manner in 3T3-L1 cells and that the ERK-dependent expression of NLRP3 may play a role independently of its canonical role as a component of inflammasomes. J. Cell. Physiol. 231: 2761-2767, 2016. © 2016 Wiley Periodicals, Inc. PMID:26989816

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

    PubMed Central

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

    2016-01-01

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

  16. Temperature homeostasis in mice lacking the p43 mitochondrial T3 receptor.

    PubMed

    Bertrand-Gaday, Christelle; Pessemesse, Laurence; Cabello, Gérard; Wrutniak-Cabello, Chantal; Casas, François

    2016-04-01

    Thyroid hormones and Thra gene play a key role in energy expenditure regulation, temperature homeostasis, and mitochondrial function. To decipher the function of the mitochondrial TRα receptor in these phenomena, we used mice lacking specifically the p43 mitochondrial T3 receptor. We found that these animals were hypermetabolic, hyperphagic, and displayed a down setting of the core body temperature. However, p43-/- animals do not present cold intolerance or defect of facultative thermogenesis. In addition, the mitochondrial function of BAT is slightly affected in the absence of p43. Our study, therefore, suggests a complementarity of action between the mitochondrial receptor and other proteins encoded by the Thra gene in the control of basal metabolism, facultative thermogenesis, and determination of the set point of temperature regulation. PMID:26970082

  17. 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. PMID:19857072

  18. 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

  19. CREB Activation Induces Adipogenesis in 3T3-L1 Cells

    PubMed Central

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

    2000-01-01

    Obesity is the result of numerous, interacting behavioral, physiological, and biochemical factors. One increasingly important factor is the generation of additional fat cells, or adipocytes, in response to excess feeding and/or large increases in body fat composition. The generation of new adipocytes is controlled by several “adipocyte-specific” transcription factors that regulate preadipocyte proliferation and adipogenesis. Generally these adipocyte-specific factors are expressed only following the induction of adipogenesis. The transcription factor(s) that are involved in initiating adipocyte differentiation have not been identified. Here we demonstrate that the transcription factor, CREB, is constitutively expressed in preadipocytes and throughout the differentiation process and that CREB is stimulated by conventional differentiation-inducing agents such as insulin, dexamethasone, and dibutyryl cAMP. Stably transfected 3T3-L1 preadipocytes were generated in which we could induce the expression of either a constitutively active CREB (VP16-CREB) or a dominant-negative CREB (KCREB). Inducible expression of VP16-CREB alone was sufficient to initiate adipogenesis as determined by triacylglycerol storage, cell morphology, and the expression of two adipocyte marker genes, peroxisome proliferator activated receptor gamma 2, and fatty acid binding protein. Alternatively, KCREB alone blocked adipogenesis in cells treated with conventional differentiation-inducing agents. These data indicate that activation of CREB was necessary and sufficient to induce adipogenesis. Finally, CREB was shown to bind to putative CRE sequences in the promoters of several adipocyte-specific genes. These data firmly establish CREB as a primary regulator of adipogenesis and suggest that CREB may play similar roles in other cells and tissues. PMID:10629058

  20. Folate receptor {alpha} regulates cell proliferation in mouse gonadotroph {alpha}T3-1 cells

    SciTech Connect

    Yao, Congjun; Evans, Chheng-Orn; Stevens, Victoria L.; Owens, Timothy R.; Oyesiku, Nelson M.

    2009-11-01

    We have previously found that the mRNA and protein levels of the folate receptor alpha (FR{alpha}) are uniquely over-expressed in clinically human nonfunctional (NF) pituitary adenomas, but the mechanistic role of FR{alpha} has not fully been determined. We investigated the effect of FR{alpha} over-expression in the mouse gonadotroph {alpha}T3-1 cell line as a model for NF pituitary adenomas. We found that the expression and function of FR{alpha} were strongly up-regulated, by Western blotting and folic acid binding assay. Furthermore, we found a higher cell growth rate, an enhanced percentage of cells in S-phase by BrdU assay, and a higher PCNA staining. These observations indicate that over-expression of FR{alpha} promotes cell proliferation. These effects were abrogated in the same {alpha}T3-1 cells when transfected with a mutant FR{alpha} cDNA that confers a dominant-negative phenotype by inhibiting folic acid binding. Finally, by real-time quantitative PCR, we found that mRNA expression of NOTCH3 was up-regulated in FR{alpha} over-expressing cells. In summary, our data suggests that FR{alpha} regulates pituitary tumor cell proliferation and mechanistically may involve the NOTCH pathway. Potentially, this finding could be exploited to develop new, innovative molecular targeted treatment for human NF pituitary adenomas.

  1. Olanzapine induces SREBP-1-related adipogenesis in 3T3-L1 cells.

    PubMed

    Yang, Li-Hung; Chen, Tzer-Ming; Yu, Sung-Tsai; Chen, Yen-Hui

    2007-09-01

    Olanzapine is a second-generation atypical antipsychotic drug (AAPD). Major side effects of olanzapine are weight gain and development of diabetes mellitus, which are risk factors of cardiovascular diseases. The possible causes of metabolic adverse effects are known as poor satiety and increased food intake due to blockade of receptors such as 5-HT(2C) in CNS. In this study, we examine the effect of olanzapine on peripheral adipogenesis using cultured 3T3-L1 cell model. Olanzapine increased triacylglyceride (TG) accumulation during 3T3-L1 preadipocyte differentiation to mature adipocyte phenotype. TG accumulation was accompanied by overexpression of fatty acid synthase and adiponectin that are the downstream genes of sterol regulatory element binding protein-1 (SREBP-1), one of the key transcription factors in lipid homeostasis. We further consisted that mostly SREBP-1 and at a lesser extent peroxisome proliferator-activated receptor gamma (PPAR-gamma), but not CCAAT/enhancer binding protein-alpha (C/EBP-alpha), were overexpressed and activated in 3T3-L1 adipocytes exposed to olanzapine. Furthermore, we showed that olanzapine enhanced the activity of SRE-1-containing LDLR promoter in transfected 3T3-L1 adipocytes and HepG2 cells. Taken together, olanzapine may cause body weight gain not only through influencing CNS receptors, but also affecting the peripheral adipogenesis regulated by SREBP-1. PMID:17651982

  2. The T3 receptor beta1 isoform regulates UCP1 and D2 deiodinase in rat brown adipocytes.

    PubMed

    Martinez de Mena, Raquel; Scanlan, Thomas S; Obregon, Maria-Jesus

    2010-10-01

    Brown adipose tissue (BAT) thermogenesis increases when uncoupling protein-1 (UCP1) is activated adrenergically and requires T3. In humans, UCP1 activation in BAT seems involved in body weight maintenance. BAT type 2 deiodinase (D2) increases in response to adrenergic agents, producing the T3 required for UCP1 expression. T3 actions are mediated by thyroid hormone nuclear T3 receptors (TR), TRα and TRβ. Studies in mice suggest that TRβ is required for UCP1 induction, whereas TRα regulates body temperature and adrenergic sensitivity. In the present study, we compare the effects of T3 vs. specific TRβ1 and TRα1 agonists [GC-1 and CO23] on the adrenergic induction of UCP1 and D2 in cultured rat brown adipocytes. T3 and GC-1 produced similar increases on UCP1, whereas CO23 increased UCP1 only at high doses (50 nm). GC-1 at low doses (0.2-10 nm) was less potent than T3, increasing the adrenergic stimulation of D2 activity and mRNA. At higher doses, GC-1 further stimulated whereas T3 inhibited D2 activity but not D2 mRNA, suggesting posttranscriptional effects. CO23 had no effect on D2 activity but increased D2 mRNA. T3, GC-1, or CO23 by themselves did not increase UCP1 or D2 mRNA. High T3 doses shortened D2 half-life and increased D2 turnover via proteasome, whereas GC-1 did not change D2 stability. The α1- and α2-adrenergic D2 responses increased using high T3 doses. In summary, T3 increases the adrenergic stimulation of UCP1 and D2 expression mostly via the TRβ1 isoform, and in brown adipocytes, D2 is protected from degradation by the action of T3 on TRβ1. PMID:20719854

  3. Mice Lacking the p43 Mitochondrial T3 Receptor Become Glucose Intolerant and Insulin Resistant during Aging

    PubMed Central

    Bertrand, Christelle; Blanchet, Emilie; Pessemesse, Laurence; Annicotte, Jean Sébastien; Feillet-Coudray, Christine; Chabi, Béatrice; Levin, Jonathan; Fajas, Lluis; Cabello, Gérard; Wrutniak-Cabello, Chantal; Casas, François

    2013-01-01

    Thyroid hormones (TH) play an important regulatory role in energy expenditure regulation and are key regulators of mitochondrial activity. We have previously identified a mitochondrial triiodothyronine (T3) receptor (p43) which acts as a mitochondrial transcription factor of the organelle genome, which leads in vitro and in vivo, to a stimulation of mitochondrial biogenesis. Recently, we generated mice carrying a specific p43 invalidation. At 2 months of age, we reported that p43 depletion in mice induced a major defect in insulin secretion both in vivo and in isolated pancreatic islets, and a loss of glucose-stimulated insulin secretion. The present study was designed to determine whether p43 invalidation influences life expectancy and modulates blood glucose and insulin levels as well as glucose tolerance or insulin sensitivity during aging. We report that from 4 months old onwards, mice lacking p43 are leaner than wild-type mice. p43−/− mice also have a moderate reduction of life expectancy compared to wild type. We found no difference in blood glucose levels, excepted at 24 months old where p43−/− mice showed a strong hyperglycemia in fasting conditions compared to controls animals. However, the loss of glucose-stimulated insulin secretion was maintained whatever the age of mice lacking p43. If up to 12 months old, glucose tolerance remained unchanged, beyond this age p43−/− mice became increasingly glucose intolerant. In addition, if up to 12 months old p43 deficient animals were more sensitive to insulin, after this age we observed a loss of this capacity, culminating in 24 months old mice with a decreased sensitivity to the hormone. In conclusion, we demonstrated that during aging the depletion of the mitochondrial T3 receptor p43 in mice progressively induced an increased glycemia in the fasted state, glucose intolerance and an insulin-resistance several features of type-2 diabetes. PMID:24098680

  4. A distinct cation-sensing mechanism in MC3T3-E1 osteoblasts functionally related to the calcium receptor.

    PubMed

    Quarles, L D; Hartle, J E; Siddhanti, S R; Guo, R; Hinson, T K

    1997-03-01

    The presence of a cation-sensing mechanism in osteoblasts is suggested by the ability of specific cations to stimulate osteoblastic proliferation in culture and to induce de novo bone formation in some experimental models. Our study examines whether extracellular cations stimulate osteoblasts through the recently identified G protein-coupled calcium receptor (CaR). We found that CaR agonists, calcium (Ca2+), gadolinium (Gd3+), aluminum (Al3+), and neomycin, stimulated DNA synthesis in murine-derived MC3T3-E1 preosteoblasts, whereas magnesium (Mg2+), nickel (Ni2+), cadmium (Cd2+), and zinc (Zn2+) had no effect. With the exception of Mg2+, the cation specificities and apparent affinities were similar to that reported for CaR. CaR agonists also stimulated DNA synthesis in C3HT10(1/2) fibroblasts, but not in mesangial PVG, CHO, hepatic HTC, COS-7 cells, or malignant transformed ROS17/2.8 and UMR-106 osteoblasts. In addition, similar to other growth factors, CaR agonists activated transcription of a serum response element luciferase reporter construct (SRE-Luc) stably transfected into MC3T3-E1 osteoblasts, but had no effect on SRE-Luc transfected into CHO and COS-7 cells. We were unable to detect CaR expression by Northern analysis using a mouse CaR-specific probe or to amplify CaR mRNA by reverse transcribed polymerase chain reaction in MC3T3-E1 osteoblasts. These findings suggest that an extra-cellular cation-sensing mechanism is present in murine-derived osteoblasts that is functionally similar to but molecularly distinct from CaR. PMID:9076582

  5. The effect of glucose concentration on insulin-induced 3T3-L1 adipose cell differentiation.

    PubMed

    Gagnon, A; Sorisky, A

    1998-03-01

    We examined the effect of glucose concentration on insulin-induced 3T3-L1 adipose cell differentiation. Oil Red O staining of neutral lipid, cellular triglyceride mass, and glycerol phosphate dehydrogenase (GPDH) activity, were greater in 3T3-L1 cells cultured at 5 mM vs. 25 mM glucose. GPDH activity was 2- to 4-fold higher at 5 mM vs. 25 mM glucose over a range of insulin concentrations (0.1 to 100 nM). Insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) was 1.7-fold greater, and insulin-stimulated phosphoinositide 3-kinase association with IRS-1 was 2.3-fold higher, at 5 mM vs. 25 mM glucose. These effects of glucose were not caused by alterations in IRS-1 mass or cell-surface insulin binding. In preadipose cells at 5 mM glucose, expression of the leukocyte antigen-related (LAR) protein tyrosine phosphatase (negative regulator of insulin signaling) was 63% of the level at 25 mM glucose. Our data demonstrate that glucose concentration affects insulin-induced 3T3-L1 adipose cell differentiation as well as differentiation-directed insulin signaling pathways. Alterations in LAR expression potentially may be involved in modulating these responses. PMID:9545023

  6. Persistent GnRH receptor activation in pituitary αT3-1 cells analyzed with a label-free technology.

    PubMed

    Nederpelt, I; Vergroesen, R D; IJzerman, A P; Heitman, L H

    2016-05-15

    The gonadotropin-releasing hormone (GnRH) receptor is a drug target for certain hormone-dependent diseases such as prostate cancer. In this study, we examined the activation profiles of the endogenous ligand, GnRH and a well-known marketed analog, buserelin using a label-free assay in pituitary αT3-1 cells with endogenous GnRH receptor expression. This whole cell impedance-based technology allows for the real-time measurement of morphological cellular changes. Both agonists dose-dependently decreased the impedance as a result of GnRH receptor activation with potencies of 9.3 ± 0.1 (pEC50 value, buserelin) and 7.8 ± 0.06 (pEC50 value, GnRH). Subsequently, GnRH receptor activation was completely abolished with a selective Gαq inhibitor, thereby confirming the Gαq-coupling of the GnRH receptor in pituitary αT3-1 cells. Additionally, we observed continued responses after agonist stimulation of αT3-1 cells indicating long-lasting cellular effects. Wash-out experiments demonstrated that the long-lasting effects induced by GnRH were most likely caused by rebinding since over 70% of the original response was abolished after wash-out. In contrast, a long receptor residence time was responsible for the prolonged effects caused by buserelin, with over 70% of the original response remaining after wash-out. In summary, we validated that impedance-based label-free technology is suited for studying receptor-mediated activation in cell lines endogenously expressing the target of interest. Moreover, this real-time monitoring allows the examination of binding kinetics and its influence on receptor activation at a cellular level. PMID:26774084

  7. 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}.

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

    SciTech Connect

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

    1988-01-12

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

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

    PubMed

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

    2016-02-01

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

  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. Temperature induced modulation of lipid oxidation and lipid accumulation in palmitate-mediated 3T3-L1 adipocytes and 3T3-L1 adipocytes.

    PubMed

    Lin, Xiaofen; Li, Yi; Leung, Polly Hangmei; Li, Jiashen; Hu, Junyan; Liu, Xuan; Li, Zhi

    2016-05-01

    Human skin temperature can vary widely depending on anatomical location and ambient temperature. It is also known that local changes in skin and subcutaneous temperature can affect fat metabolism. This study aimed to explore the potential effects of surrounding thermal environment on fat by investigating cell viability, lipid oxidation, and lipid accumulation in 3T3-L1 adipocytes and palmitate-treated adipocytes after 4h incubation. No significant differences of viability in 3T3-L1 adipocytes were detected under different temperature conditions. Despite no significant increase being observed under warm temperature (39°C) conditions, a similarly significant suppression of intracellular reactive oxygen species (ROS) and lipid peroxidation were found in 3T3-L1 adipocytes and palmitate-treated adipocytes under 4h exposure to cooler temperatures of 31-33°C (P<0.01). ROS, chemically reactive molecules containing oxygen, are currently understood to be a major contributor to oxidantive stress in obesity. Additionally, cooler temperatures (31-33°C) could improve the size of lipid droplets in 3T3-L1 adipocytes (P<0.01), but no significant effect was generated by temperature change on lipid droplets in palmitate-treated adipocytes. In the palmitate-induced adiposity model, although excessive ROS and lipid peroxidation has been attenuated by temperature decrease (P<0.01), it still does not positively modulate lipid droplet size (P>0.05) and remedy the palmitate damage induced cell death (P<0.01). These findings provide preliminary support for potential interventions based on temperature manipulation for cell metabolism of adipocytes. PMID:27157327

  12. Changes in laser-induced fluorescence responses of 3T3 fibroblasts to repetitive thermal stress

    NASA Astrophysics Data System (ADS)

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

    2009-04-01

    The combined experimental use of laser-induced autofluorescence of cellular metabolites and methodological fundamentals of systems biology will provide access to biological thermal stress analysis on a sub cellular level. A test setup incorporating a pulsed nitrogen laser was realized with which autofluorescence of the coenzyme NADH could be measured in living 3T3 cells. The cells were subjected to different temperature stress at repetitive time intervals. When subjected to a simple mathematical analysis, the NADH concentration change measured through autofluorescence in biological cells exhibited approximate concentration-equivalent balance curves. These results add up to the fundamental know-how about the dosimetry of thermally therapeutic methods.

  13. 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

  14. 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. PMID:26896766

  15. The influence of triiodothyronine (T3) and inducers on hepatic drug metabolism in rats of different ages.

    PubMed

    Müller, D; Greiling, K; Greiling, H; Klinger, W

    1985-01-01

    After a 3-day treatment with T3 the ethylmorphine N-demethylation rate was diminished in 10- to 60-day-old rats, whereas the ethoxycoumarin O-deethylation rate was distinctly enhanced in some age-groups, particularly in 33-day-old rats. P-450 concentration was decreased in young rats and not changed in 60-day-old ones. Phenobarbital administration enhanced the rate of both reactions and P-450 concentration in all age-groups. This treatment significantly attenuated the T3 effects on P-450 concentration and ethylmorphine N-demethylation. beta-Naphthoflavone induced ethoxycoumarin O-deethylation and P-450 concentration in all age-groups. Compared with non-induced animals, the T3 effects on ethoxycoumarin O-deethylation (increase) and ethylmorphine N-demethylation (decrease) were more distinct, whereas P-450 concentration was scarcely influenced by T3. Age-differences in T3 actions were generally diminished by inducers. PMID:4084270

  16. Influence of triiodothyronine (T(3)) on secretion of steroids and thyroid hormone receptor expression in chicken ovarian follicles.

    PubMed

    Sechman, A; Pawlowska, K; Rzasa, J

    2009-08-01

    The present study was designed to (1) assess the role of triiodothyronine (T(3)) with regard to in vitro steroid hormone secretion by chicken ovarian follicles; (2) determine whether T(3) influences the in vivo function of the pituitary-ovarian axis in the hen; and (3) detect expression of thyroid hormone receptor (TR) mRNA in chicken ovarian follicles. In the first experiment, laying hens were decapitated 22.5h before ovulation. White prehierarchical follicles (1-8mm) and fragments of theca and granulosa layers of the 3 largest yellow preovulatory follicles F3-F1 (22-35mm) were incubated in a medium supplemented with T(3) (0, 0.1, 1, 10, 100, or 1000ng/mL) or ovine luteinizing hormone (LH) (10ng/mL) in combination with doses of T(3) (1, 10, and 100ng/mL). Triiodothyronine decreased basal and LH-stimulated estradiol secretion by white follicles and the theca layer of all preovulatory follicles. On the other hand, it increased progesterone secretion by F2 and F1 follicles. In the second experiment, hens were injected 1h after ovulation with saline (control) or T(3) (10microg/100g body weight, intraperitoneally). Results indicated that exogenous T(3) decreased plasma concentrations of LH and estradiol and increased plasma concentrations of progesterone. In the third experiment, using reverse transcription polymerase chain reaction (RT-PCR) analysis, expression of thyroid hormone receptor (TRalpha and TRbeta0), mRNA was detected in all of the ovarian compartments. The expression of TRalpha mRNA was relatively greater in comparison with TRbeta0. There were no differences between white ovarian follicles in the expression of TRalpha and TRbeta0 mRNA. A considerably higher TRalpha and lower TRbeta0 expression was detected in the granulosa layer of preovulatory follicles in comparison with the theca layer. In conclusion, the data indicate that thyroid hormones acting via nuclear receptors are involved in regulation of the pituitary-ovarian axis and processes associated

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

    PubMed Central

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

    1994-01-01

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

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

    SciTech Connect

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

    2005-05-01

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

  19. Protective effect of quercitrin against hydrogen peroxide-induced dysfunction in osteoblastic MC3T3-E1 cells.

    PubMed

    Choi, Eun Mi

    2012-03-01

    The protective effect of quercitrin on the response of osteoblastic MC3T3-E1 cells to oxidative stress was evaluated. Osteoblasts were incubated with H(2)O(2) and/or quercitrin, and markers of osteoblast function and oxidative damage were examined. Quercitrin treatment reversed the cytotoxic effect of H(2)O(2) significantly (P<0.05). This effect was blocked by ICI182780 and LY294002, suggesting that quercitrin's effect might be involved in estrogen action and results from PI3K mediated signaling pathway. Pretreatment of quercitrin increased collagen content, alkaline phosphatase (ALP) activity, and calcium deposition of osteoblasts compared with H(2)O(2) treated cells and these effects were blocked by ERKs and p38 mitogen-activated protein kinases (MAPKs) inhibitors such as PD98059 and SB203580, respectively. These suggest that quercitrin-induced protective effect against osteoblast dysfunction by oxidative stress is associated with increased activation of ERKs and p38 MAPK. Pretreatment with quercitrin also reduced the increase in bone-resorbing factor, receptor activator of nuclear factor-kB ligand (RANKL) and oxidative damage markers (malondialdehyde, protein carbonyl, and nitrotyrosine) induced by H(2)O(2). These results suggest that quercitrin may be protective against H(2)O(2)-induced dysfunction in osteoblasts. PMID:20822887

  20. Expression of an exogenous eukaryotic DNA methyltransferase gene induces transformation of NIH 3T3 cells.

    PubMed Central

    Wu, J; Issa, J P; Herman, J; Bassett, D E; Nelkin, B D; Baylin, S B

    1993-01-01

    Abnormal regional increases in DNA methylation, which have potential for causing gene inactivation and chromosomal instability, are consistently found in immortalized and tumorigenic cells. Increased DNA methyltransferase activity, which is also a characteristic of such cells, is a candidate to mediate these abnormal DNA methylation patterns. We now show that, in NIH 3T3 mouse fibroblasts, constitutive overexpression of an exogenous mouse DNA methyltransferase gene results in a marked increase in overall DNA methylation which is accompanied by tumorigenic transformation. These transformation changes can also be elicited by dexamethasone-inducible expression of an exogenous DNA methyltransferase gene. Our findings provide strong evidence that the increase in DNA methyltransferase activity associated with tumor progression could be a key step in carcinogenesis and provide a model system that can be used to further study this possibility. Images Fig. 1 Fig. 2 PMID:8415627

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

    SciTech Connect

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

    1994-12-31

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

  2. HeLa cell response proteome alterations induced by mammalian reovirus T3D infection

    PubMed Central

    2013-01-01

    Background Cells are exposed to multiple stressors that induce significant alterations in signaling pathways and in the cellular state. As obligate parasites, all viruses require host cell material and machinery for replication. Virus infection is a major stressor leading to numerous induced modifications. Previous gene array studies have measured infected cellular transcriptomes. More recently, mass spectrometry-based quantitative and comparative assays have been used to complement such studies by examining virus-induced alterations in the cellular proteome. Methods We used SILAC (stable isotope labeling with amino acids in cell culture), a non-biased quantitative proteomic labeling technique, combined with 2-D HPLC/mass spectrometry and reciprocal labeling to identify and measure relative quantitative differences in HeLa cell proteins in purified cytosolic and nuclear fractions after reovirus serotype 3 Dearing infection. Protein regulation was determined by z-score analysis of each protein’s label distribution. Results A total of 2856 cellular proteins were identified in cytosolic fractions by 2 or more peptides at >99% confidence and 884 proteins were identified in nuclear fractions. Gene ontology analyses indicated up-regulated host proteins were associated with defense responses, immune responses, macromolecular binding, regulation of immune effector processes, and responses to virus, whereas down-regulated proteins were involved in cell death, macromolecular catabolic processes, and tissue development. Conclusions These analyses identified numerous host proteins significantly affected by reovirus T3D infection. These proteins map to numerous inflammatory and innate immune pathways, and provide the starting point for more detailed kinetic studies and delineation of virus-modulated host signaling pathways. PMID:23799967

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

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

  4. Characterization of a bombesin receptor on Swiss mouse 3T3 cells by affinity cross-linking

    SciTech Connect

    Sinnett-Smith, J.; Zachary, I.; Rozengurt, E.

    1988-12-01

    We have previously identified by chemical cross-linking a cell surface protein in Swiss 3T3 cells of apparent Mr 75,000-85,000, which may represent a major component of the receptor for peptides of the bombesin family in these cells. Because bombesin-like peptides may interact with other cell surface molecules, it was important to establish the correlation between receptor binding and functions of this complex and further characterize the Mr 75,000-85,000 cross-linked protein. Detailed time courses carried out at different temperatures demonstrated that the Mr 75,000-85,000 affinity-labelled band was the earliest cross-linked complex detected in Swiss 3T3 cells incubated with 125I-labelled gastrin-releasing peptide (125I-GRP). Furthermore, the ability of various nonradioactive bombesin agonists and antagonists to block the formation of the Mr 75,000-85,000 cross-linked complex correlated extremely well (r = 0.994) with the relative capacity of these peptides to inhibit 125I-GRP specific binding. Pretreatment with unlabelled GRP for up to 6 h caused only a slight decrease in both specific 125I-GRP binding and the affinity labelling of the Mr 75,000-85,000 protein. We also show that the cross-linked complex is a glycoprotein. First, solubilized affinity labelled Mr 75,000-85,000 complex applied to wheat germ lectin-sepharose columns was eluted by addition of 0.3 M N-acetyl-D-glucosamine. Second, treatment with endo-beta-N-acetylglucosaminidase F reduced the apparent molecular weight of the affinity-labelled band from 75,000-85,000 to 43,000, indicating the presence of N-linked oligosaccharide groups.

  5. 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

  6. Epidermal growth factor-nonresponsive 3T3 variants do not contain epidermal growth factor receptor-related antigens or mRNA

    SciTech Connect

    Schneider, C.A.; Lim, R.W.; Terwilliger, E.; Herschman, H.R.

    1986-01-01

    The authors have previously isolated three independent variants of Swiss 3T3 cells that are unable to generate a mitogenic response to epidermal growth factor (EGF). Each of the variants is unable to bind /sup 125/I-labeled EGF; each lacks a functional EGF receptor. They used an antiserum to murine EGF receptor to look for an EGF-receptor gene product in wild-type 3T3 cells and in the three EGF-nonresponsive variants. No cross-reactive material could be detected in any of the three variants, either in /sup 125/I-labeled cell extracts or in (/sup 35/S)methionine metabolically labeled cells. 3T3 cells contained mRNA molecules homologous to a cDNA probe for the human EGF-receptor coding region. In contrast, no homologous RNA could be detected in any of the three variants. Analysis of genomic Southern blots of the DNA from 3T3 cells and the three EGF-nonresponsive variants indicated sequences from the EGF-receptor gene are present in the DNA of all four cell lines. These EGF-nonresponsive lines, which demonstrate proliferative responses to a variety of mitogens, will be ideal recipients for structure-function studies of the EGF receptor by transfection of the cloned gene.

  7. High glucose induces autophagy of MC3T3-E1 cells via ROS-AKT-mTOR axis.

    PubMed

    Wang, Xiaoju; Feng, Zhengping; Li, Jiling; Chen, Lixue; Tang, Weixue

    2016-07-01

    In the present study, we investigate the function of ROS-AKT-mTOR axis on the apoptosis, proliferation and autophagy of MC3T3-E1 cells, and the proliferation of MC3T3-E1 cells after autophagy inhibition under high glucose conditions. MC3T3-E1 cells cultured in vitro were divided into the following groups: normal control group, N-acetylcysteine (NAC) group, 11.0 mM high glucose group, 11.0 mM high glucose + NAC group, 22.0 mM high glucose group, 22.0 mM high glucose + NAC group, CQ group, 22.0 mM high glucose + CQ group, 3-MA group and 3-MA + 22.0 mM high glucose group. ROS production was measured by DCFH-DA fluorescent probe. Cell proliferation was measured by MTT assay. Cells in different groups were stained with Annexin V-FITC/PI, and then apoptosis rate was detected by flow cytometry. Nucleus morphology was observed under fluorescence microscope after being incubated with Honchest33258. Protein expression was measured using Western blotting and immunofluorescence. Cell apoptosis and proliferation in high glucose group were increased and decreased, respectively, in a dose-dependent manner. Autophagy was significantly induced in high glucose group, even though different concentration of glucose induced autophagy in different stages of autophagy. ROS production in MC3T3-E1 cells was remarkably increased in high glucose group, but not in a dose-dependent manner. NAC, as an antioxidant, reduced ROS production and ameliorated cell apoptosis, proliferation abnormity and autophagy caused by high glucose. Expression of p-AKT and p-mTOR proteins were dramatically decreased in high glucose group, and NAC reversed their expression. In addition, 3-MA, an inhibitor of autophagy, significantly decreased the proliferation of MC3T3-E1 cells. When cocultured with 22.0 mM glucose that induced autophagy, proliferation of MC3T3-E1 cells was not affected compared to 22.0 mM high glucose group. Our present findings reveal that high glucose affects apoptosis

  8. Uric acid induces oxidative stress via an activation of the renin-angiotensin system in 3T3-L1 adipocytes.

    PubMed

    Zhang, Jun-xia; Zhang, Yu-ping; Wu, Qi-nan; Chen, Bing

    2015-02-01

    Hyperuricemia is recently reported involving in various obesity-related cardiovascular disorders, especially hypertension. However, the underlying mechanisms are not completely understood. In the present study, we investigated whether uric acid upregulates renin-angiotensin system (RAS) expression in adipocytes. We also examined whether RAS activation plays a role in uric acid-induced oxidative stress in adipocytes. The adipocytes of different phenotypes were incubated with uric acid for 48 h, respectively. Losartan (10(-4) M) or captopril (10(-4) M) was used to block adipose tissue RAS activation. mRNA expressions of angiotensinogen (AGT), angiotensin-converting enzyme-1 (ACE-1), renin, angiotensin type 1 receptor (AT1R), and angiotensin type 2 receptor (AT2R) were evaluated with real-time PCR. Angiotensin II concentrations in supernatant were measured by ELISA. Intracellular reactive species (ROS) levels were measured by fluorescent probe DCFH-DA, DHR, or NBT assay. The uric acid upregulated both RAS (AGT, ACE1, renin, AT1R, and AT2R) mRNA expressions and angiotensin II protein secretion and caused a significant increase in ROS production in 3T3-L1 adipocytes. These effects could be prevented by RAS inhibitors, either losartan or captopril. RAS activation has been causally implicated in oxidative stress induced by uric acid in 3T3-L1 adipocytes, suggesting a plausible mechanism through which hyperuricemia contributes to the pathogenesis of obesity-related cardiovascular diseases. PMID:24671741

  9. 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.

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

    PubMed

    Choi, Eun Mi

    2013-06-01

    Deoxyactein is one of the major constituents isolated from Cimicifuga racemosa. In the present study, we investigated the protective effects of deoxyactein on antimycin A (mitochondrial electron transport inhibitor)-induced toxicity in osteoblastic MC3T3-E1 cells. Exposure of MC3T3-E1 cells to antimycin A caused significant cell viability loss, as well as mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, intracellular calcium ([Ca(2+) ]i ) elevation and oxidative stress. Pretreatment with deoxyactein prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, [Ca(2+) ]i elevation and oxidative stress. Moreover, deoxyactein increased the activation of PI3K (phosphoinositide 3-kinase), Akt (protein kinase B) and CREB (cAMP-response element-binding protein) inhibited by antimycin A. All these data indicate that deoxyactein may reduce or prevent osteoblasts degeneration in osteoporosis or other degenerative disorders. PMID:22180388

  11. Synthesis of membrane-bound colony-stimulating factor 1 (CSF-1) and downmodulation of CSF-1 receptors in NIH 3T3 cells transformed by cotransfection of the human CSF-1 and c-fms (CSF-1 receptor) genes.

    PubMed Central

    Rettenmier, C W; Roussel, M F; Ashmun, R A; Ralph, P; Price, K; Sherr, C J

    1987-01-01

    NIH 3T3 cells cotransfected with the human c-fms proto-oncogene together with a 1.6-kilobase cDNA clone encoding a 256-amino-acid precursor of the human mononuclear phagocyte colony-stimulating factor CSF-1 (M-CSF) undergo transformation by an autocrine mechanism. The number of CSF-1 receptors on the surface of transformed cells was regulated by ligand-induced receptor degradation and was inversely proportional to the quantity of CSF-1 produced. A tyrosine-to-phenylalanine mutation at position 969 near the receptor carboxyl terminus potentiated its transforming efficiency in cells cotransfected by the CSF-1 gene but did not affect receptor downmodulation. CSF-1 was synthesized as an integral transmembrane glycoprotein that was rapidly dimerized through disulfide bonds. The homodimer was externalized at the cell surface, where it underwent proteolysis to yield the soluble growth factor. Trypsin treatment of viable cells cleaved the plasma membrane form of CSF-1 to molecules of a size indistinguishable from that of the extracellular growth factor, suggesting that trypsinlike proteases regulate the rate of CSF-1 release from transformed cells. The data raise the possibility that this form of membrane-bound CSF-1 might stimulate receptors on adjacent cells through direct cell-cell interactions. Images PMID:3039346

  12. 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

  13. Bezafibrate prevents palmitate-induced apoptosis in osteoblastic MC3T3-E1 cells through the NF-κB signaling pathway.

    PubMed

    Zhong, Xing; Xiu, Lingling; Wei, Guohong; Pan, Tianrong; Liu, Yuanyuan; Su, Lei; Li, Yanbing; Xiao, Haipeng

    2011-10-01

    Osteoporosis is a bone condition defined by low bone mass and increase of fracture risk due to imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Low bone mass is likely to be due to the alteration of the osteoclast and osteoblast lifespan through regulated apoptosis. Saturated fatty acid (SFA) intake is negatively associated with bone mineral density (BMD). Furthermore, SFA induces apoptosis in osteoblastic cell lines. Bezafibrate could increase bone mass in intact male rats principally through increasing periosteal bone formation. At present, it is unknown whether bezafibrate attenuates palmitate-induced apoptosis in MC3T3-E1 cells. In the present study, we found that palmitate stimulated the degradation of IκBα and NF-κB translocation, as well as up-regulation of NF-κB-mediated Fas expression in obsteoblastic MC3T3-E1 cells. Furthermore, the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) could restore palmitate-induced caspase-3 decrease and inhibit palmitate-induced cleaved caspase-3 increase. We observed that bezafibrate, a dual ligand for the peroxisome proliferator-activated receptors α (PPARα) and PPARδ, significantly attenuated the palmitate-induced cytotoxicity as determined by the MTT assay and inhibited the palmitate-induced apoptosis as determined by a flow cytometry assay using Annexin V-FITC/PI and assessment of the activity of caspase-3. Pre-treatment of bezafibrate prevented palmitate-induced NF-κB activation. Therefore, these findings indicate that bezafibrate inbibits palmitate-induced apoptosis via the NF-κB signaling pathway. Our results point to bezafibrate as a new strategy to attenuate bone loss associated with high fat diet beyond its lipid-lowering actions. PMID:21687928

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

    PubMed

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

    2015-03-01

    To investigate the potential adverse effects of mobile phone radiation, we studied reactive oxygen species (ROS), DNA damage and apoptosis in mouse embryonic fibroblasts (NIH/3T3) after intermittent exposure (5 min on/10 min off, for various durations from 0.5 to 8 h) to an 1800-MHz GSM-talk mode electromagnetic radiation (EMR) at an average specific absorption rate of 2 W/kg. A 2',7'-dichlorofluorescin diacetate fluorescence probe was used to detect intracellular ROS levels, immunofluorescence was used to detect γH2AX foci as a marker for DNA damage, and flow cytometry was used to measure apoptosis. Our results showed a significant increase in intracellular ROS levels after EMR exposure and it reached the highest level at an exposure time of 1 h (p < 0.05) followed by a slight decrease when the exposure continued for as long as 8 h. No significant effect on the number of γH2AX was detected after EMR exposure. The percentage of late-apoptotic cells in the EMR-exposed group was significantly higher than that in the sham-exposed groups (p < 0.05). These results indicate that an 1800-MHz EMR enhances ROS formation and promotes apoptosis in NIH/3T3 cells. PMID:24665905

  15. RA induces the neural-like cells generated from epigenetic modified NIH/3T3 cells.

    PubMed

    Zhang, Xi-Mei; Li, Qiu-Ming; Su, Dong-Ju; Wang, Ning; Shan, Zhi-Yan; Jin, Lian-Hong; Lei, Lei

    2010-03-01

    Recently, differentiated somatic cells had been reprogrammed to pluripotential state in vitro, and various tissue cells had been elicited from those cells. Epigenetic modifications allow differentiated cells to perpetuate the molecular memory needed for the cells to retain their identity. DNA methylation and histone deacetylation are important patterns involved in epigenetic modification, which take critical roles in regulating DNA expression. In this study, we dedifferentiated NIH/3T3 fibroblasts by 5-aza-2-deoxycytidine (5-aza-dC) and Trichstatin A (TSA) combination, and detected gene expression pattern, DNA methylation level, and differentiation potential of reprogrammed cells. As the results, embryonic marker Sox2, klf4, c-Myc and Oct4 were expressed in reprogrammed NIH/3T3 fibroblasts. Total DNA methylation level was significant decreased after the treatment. Moreover, exposure of the reprogrammed cells to all trans-retinoic acid (RA) medium elicited the generation of neuronal class IIIbeta-tubulin-positive, neuron-specific enolase (NSE)-positive, nestin-positive, and neurofilament light chain (NF-L)-positive neural-like cells. PMID:19263240

  16. Functional proteomic analysis of long-term growth factor stimulation and receptor tyrosine kinase coactivation in Swiss 3T3 fibroblasts.

    PubMed

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

    2012-12-01

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

  17. Zinc deficiency induced in Swiss 3T3 cells by a low-zinc medium impairs calcium entry and two mechanisms of entry are involved.

    PubMed

    O'Dell, Boyd L; Browning, Jimmy D

    2013-04-01

    Zinc deficiency in 3T3 cells induced by the use of diethylenetriaminepentaacetate (DTPA) has been shown to impair calcium entry associated with failure of proliferation when the cells are stimulated with polypeptide growth factors (GF). These functions of zinc have been evaluated here in the same clone of cells by simple depletion using a low-zinc medium (0.05 μmol/L zinc) without chelator. Confluent cells were maintained for 1 day in the low-zinc medium without GF, then loaded with Fluo-4, and stimulated with GF. Calcium entry was measured by the increase in sustained fluorescence. It was preceded by the release of stored calcium as observed in the previous study using DTPA. Zinc deprivation decreased calcium entry when calcium was added at 0 or 0.05 mmol/L but not when 0.1 mmol/L or higher. Cell proliferation reflected similar effects of zinc and calcium concentrations. In a newly acquired clone of 3T3 cells, GF did not induce internal calcium release but thapsigargin (TG) did. When added in a low-calcium medium, both agonists stimulated calcium entry when external calcium was added, suggesting that two different mechanisms of entry were impaired by zinc deficiency. Zinc deficiency produced by DTPA in the newer clones gave similar results, decreasing calcium entry induced by both agonists. The effects of GF and TG were not additive. The results confirm the earlier observation that zinc deficiency impairs calcium entry into 3T3 cells when stimulated by GF and show that the cells can take up calcium by either store-operated or receptor-operated mechanisms. PMID:23292302

  18. Osmotically inducible uptake of betaine via amino acid transport system A in SV-3T3 cells.

    PubMed

    Petronini, P G; De Angelis, E; Borghetti, A F; Wheeler, K P

    1994-05-15

    The osmotically inducible uptake of betaine (NNN-trimethylglycine) by SV-3T3 cells has been studied and compared with the similar process in MDCK cells. Betaine uptake by SV-3T3 cells could be described in terms of a saturable, Na(+)-dependent, component plus a small non-saturable, Na(+)-independent, component. Transport was active, producing considerable accumulation of betaine in the cells. After exposure of the cells to hypertonic conditions for 6 h, there was a marked increase in betaine uptake. Kinetic analysis indicated that this increase resulted from an increase in the Vmax. value of the saturable component, from about 88 to 185 nmol of betaine/5 min per mg of protein, the corresponding Km values of about 15 and 10 mM not being significantly different. This induction of transport activity was detectable only after about 2 h exposure of the cells to hypertonic medium, closely paralleling an induction of influx of N-methylaminoisobutyric acid, and was prevented by the presence of cycloheximide. Betaine influx was markedly inhibited by several neutral amino acids, particularly those transported by system A, such as N-methylaminoisobutyric acid and the imino acid proline. A high concentration (25 mM) of betaine also significantly inhibited the uptake of proline by SV-3T3 cells. Although very similar results were obtained with MDCK cells, prolonged exposure of cells to hypertonic conditions revealed distinct differences. When the hypertonic incubation was extended from 6 h to 24 h, betaine transport in SV-3T3 cells either remained the same or decreased, whereas it showed a further marked increase in MDCK cells, and also became sensitive to inhibition by gamma-aminobutyric acid. mRNA for the betaine transporter BGT-1 [Yamauchi, Uchida, Kwon, Preston, Brooks Robey, Garcia-Perez, Burg and Handler (1992) J. Biol. Chem. 267, 649-652] was detectable in MDCK cells exposed to hypertonic medium for 24 h, but not in SV-3T3 cells under any conditions. It is concluded that

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

    PubMed

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

    2015-12-01

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

  20. Labisia pumila Upregulates Peroxisome Proliferator-Activated Receptor Gamma Expression in Rat Adipose Tissues and 3T3-L1 Adipocytes

    PubMed Central

    Gu, Harvest F.; Östenson, Claes-Göran; Mannerås-Holm, Louise; Stener-Victorin, Elisabet; Wan Mohamud, Wan Nazaimoon

    2013-01-01

    Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-activated transcription factor that regulates lipid and glucose metabolism. We investigated the effects of Labisia pumila (LP) standardized water extract on PPARgamma transcriptional activity in adipocytes in vitro and in vivo. We used a rat model of dihydrotestosterone- (DHT-) induced polycystic ovary syndrome (PCOS), a condition characterized by insulin resistance. At 9 weeks of age, the PCOS rats were randomly subdivided into two groups: PCOS-LP (50 mg/kg/day of LP) and PCOS-control (1 mL of deionised water) for 4-5 weeks on the same schedule. Real-time RT-PCR was performed to determine the PPARgamma mRNA levels. LP upregulated PPARgamma mRNA level by 40% in the PCOS rats. Western blot analysis further demonstrated the increased PPARgamma protein levels in parallel with upregulation in mRNA. These observations were further proven by adipocytes culture. Differentiated 3T3-L1 adipocytes were treated with final concentration of 100 μg/mL LP and compared to untreated control and 10 μM of rosiglitazone (in type of thiazolidinediones). LP increased PPARgamma expressions at both mRNA and protein levels and enhanced the effect of glucose uptake in the insulin-resistant cells. The data suggest that LP may ameliorate insulin resistance in adipocytes via the upregulation of PPARgamma pathway. PMID:23935612

  1. Labisia pumila Upregulates Peroxisome Proliferator-Activated Receptor Gamma Expression in Rat Adipose Tissues and 3T3-L1 Adipocytes.

    PubMed

    Mansor, Fazliana; Gu, Harvest F; Ostenson, Claes-Göran; Mannerås-Holm, Louise; Stener-Victorin, Elisabet; Wan Mohamud, Wan Nazaimoon

    2013-01-01

    Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-activated transcription factor that regulates lipid and glucose metabolism. We investigated the effects of Labisia pumila (LP) standardized water extract on PPARgamma transcriptional activity in adipocytes in vitro and in vivo. We used a rat model of dihydrotestosterone- (DHT-) induced polycystic ovary syndrome (PCOS), a condition characterized by insulin resistance. At 9 weeks of age, the PCOS rats were randomly subdivided into two groups: PCOS-LP (50 mg/kg/day of LP) and PCOS-control (1 mL of deionised water) for 4-5 weeks on the same schedule. Real-time RT-PCR was performed to determine the PPARgamma mRNA levels. LP upregulated PPARgamma mRNA level by 40% in the PCOS rats. Western blot analysis further demonstrated the increased PPARgamma protein levels in parallel with upregulation in mRNA. These observations were further proven by adipocytes culture. Differentiated 3T3-L1 adipocytes were treated with final concentration of 100  μ g/mL LP and compared to untreated control and 10  μ M of rosiglitazone (in type of thiazolidinediones). LP increased PPARgamma expressions at both mRNA and protein levels and enhanced the effect of glucose uptake in the insulin-resistant cells. The data suggest that LP may ameliorate insulin resistance in adipocytes via the upregulation of PPARgamma pathway. PMID:23935612

  2. RELAXIN enhances differentiation and matrix mineralization through Relaxin/insulin-like family peptide receptor 2 (Rxfp2) in MC3T3-E1 cells in vitro.

    PubMed

    Duarte, Carolina; Kobayashi, Yukiho; Kawamoto, Tatsuo; Moriyama, Keiji

    2014-08-01

    RELAXIN (RLN) is a polypeptide hormone of the insulin-like hormone family; it facilitates birth by softening and widening the pubic symphysis and cervix in many mammals, including humans. The role of RLN in bone metabolism was recently suggested by its ability to induce osteoclastogenesis and activate osteoclast function. RLN binds to RELAXIN/INSULIN-LIKE FAMILY PEPTIDE 1 (RXFP1) and 2 (RXFP2), with varying species-specific affinities. Young men with mutated RXFP2 are at high risk for osteoporosis, as RXFP2 influences osteoblast metabolism by binding to INSULIN-LIKE PEPTIDE 3 (INSL3). However, there have been no reports on RLN function in osteoblast differentiation and mineralization or on the functionally dominant receptors for RLN in osteoblasts. We previously described Rxfp1 and 2 expression patterns in developing mouse oral components, including the maxillary and mandibular bones, Meckel's cartilage, tongue, and tooth primordia. We hypothesized that Rln/Rxfp signaling is a key mediator of skeletal development and metabolism. Here, we present the gene expression patterns of Rxfp1 and 2 in developing mouse calvarial frontal bones as determined by in situ hybridization. In addition, RLN enhanced osteoblastic differentiation and caused abnormal mineralization and extracellular matrix metabolism through Rxfp2, which was predominant over Rxfp1 in MC3T3-E1 mouse calvarial osteoblasts. Our data suggest a novel role for Rln in craniofacial skeletal development and metabolism through Rxfp2. PMID:24857857

  3. 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

  4. Trichostatin A modulates thiazolidinedione-mediated suppression of tumor necrosis factor α-induced lipolysis in 3T3-L1 adipocytes.

    PubMed

    Lu, Juu-Chin; Chang, Yu-Tzu; Wang, Chih-Tien; Lin, Yu-Chun; Lin, Chun-Ken; Wu, Zhong-Sheng

    2013-01-01

    In obesity, high levels of tumor necrosis factor α (TNFα) stimulate lipolysis in adipocytes, leading to hyperlipidemia and insulin resistance. Thiazolidinediones (TZDs), the insulin-sensitizing drugs, antagonize TNFα-induced lipolysis in adipocytes, thereby increasing insulin sensitivity in diabetes patients. The cellular target of TZDs is peroxisome proliferator-activated receptor γ (PPARγ), a nuclear receptor that controls many adipocyte functions. As a transcription factor, PPARγ is closely modulated by coregulators, which include coactivators and corepressors. Previous studies have revealed that in macrophages, the insulin-sensitizing effect of PPARγ may involve suppression of proinflammatory gene expression by recruiting the corepressor complex that contains corepressors and histone deacetylases (HDACs). Therefore, we investigated whether the corepressor complex is involved in TZD-mediated suppression of TNFα-induced lipolysis in 3T3-L1 adipocytes. Trichostatin A (TSA), a pan HDAC inhibitor (HDACI) that inhibits class I and II HDACs, was used to examine the involvement of HDACs in the actions of TZDs. TSA alone increased basal lipolysis and attenuated TZD-mediated suppression of TNFα-induced lipolysis. Increased basal lipolysis may in part result from class I HDAC inhibition because selective class I HDACI treatment had similar results. However, attenuation of TZD-mediated TNFα antagonism may be specific to TSA and related hydroxamate-based HDACI rather than to HDAC inhibition. Consistently, corepressor depletion did not affect TZD-mediated suppression. Interestingly, TSA treatment greatly reduced PPARγ levels in differentiated adipocytes. Finally, extracellular signal-related kinase 1/2 (ERK1/2) mediated TNFα-induced lipolysis, and TZDs suppressed TNFα-induced ERK phosphorylation. We determined that TSA increased basal ERK phosphorylation, and attenuated TZD-mediated suppression of TNFα-induced ERK phosphorylation, consistent with TSA's effects

  5. A Nanodot Array Modulates Cell Adhesion and Induces an Apoptosis-Like Abnormality in NIH-3T3 Cells

    NASA Astrophysics Data System (ADS)

    Pan, Hsu-An; Hung, Yao-Ching; Su, Chia-Wei; Tai, Shih-Ming; Chen, Chiun-Hsun; Ko, Fu-Hsiang; Steve Huang, G.

    2009-08-01

    Micro-structures that mimic the extracellular substratum promote cell growth and differentiation, while the cellular reaction to a nanostructure is poorly defined. To evaluate the cellular response to a nanoscaled surface, NIH 3T3 cells were grown on nanodot arrays with dot diameters ranging from 10 to 200 nm. The nanodot arrays were fabricated by AAO processing on TaN-coated wafers. A thin layer of platinum, 5 nm in thickness, was sputtered onto the structure to improve biocompatibility. The cells grew normally on the 10-nm array and on flat surfaces. However, 50-nm, 100-nm, and 200-nm nanodot arrays induced apoptosis-like events. Abnormality was triggered after as few as 24 h of incubation on a 200-nm dot array. For cells grown on the 50-nm array, the abnormality started after 72 h of incubation. The number of filopodia extended from the cell bodies was lower for the abnormal cells. Immunostaining using antibodies against vinculin and actin filament was performed. Both the number of focal adhesions and the amount of cytoskeleton were decreased in cells grown on the 100-nm and 200-nm arrays. Pre-coatings of fibronectin (FN) or type I collagen promoted cellular anchorage and prevented the nanotopography-induced programed cell death. In summary, nanotopography, in the form of nanodot arrays, induced an apoptosis-like abnormality for cultured NIH 3T3 cells. The occurrence of the abnormality was mediated by the formation of focal adhesions.

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

    NASA Astrophysics Data System (ADS)

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

    2010-04-01

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

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

    PubMed

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

    2000-07-26

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

  8. Green Tea (-)-Epigallotocatechin-3-Gallate Induces PGC-1α Gene Expression in HepG2 Cells and 3T3-L1 Adipocytes.

    PubMed

    Lee, Mak-Soon; Lee, Seohyun; Doo, Miae; Kim, Yangha

    2016-03-01

    Green tea (Camellia sinensis) is one of the most popular beverages in the world and has been acknowledged for centuries as having significant health benefits. (-)-Epigallocatechin-3-gallate (EGCG) is the most abundant catechin in green tea, and it has been reported to have health benefit effects. Peroxisome proliferator-activated receptor γ coactivator (PGC)-1α is a crucial regulator of mitochondrial biogenesis and hepatic gluconeogenesis. The objective of this study was to investigate whether EGCG from green tea can affect the ability of transcriptional regulation on PGC-1α mRNA expression in HepG2 cells and 3T3-L1 adipocytes. To study the molecular mechanism that allows EGCG to control PGC-1α expression, the promoter activity levels of PGC-1α were examined. The PGC-1α mRNA level was measured using quantitative real-time PCR. The -970/+412 bp of PGC-1α promoter was subcloned into the pGL3-Basic vector that includes luciferase as a reporter gene. EGCG was found to up-regulate the PGC-1α mRNA levels significantly with 10 μmol/L of EGCG in HepG2 cells and differentiated 3T3-L1 adipocytes. PGC-1α promoter activity was also increased by treatment with 10 μmol/L of EGCG in both cells. These results suggest that EGCG may induce PGC-1α gene expression, potentially through promoter activation. PMID:27069908

  9. Green Tea (−)-Epigallotocatechin-3-Gallate Induces PGC-1α Gene Expression in HepG2 Cells and 3T3-L1 Adipocytes

    PubMed Central

    Lee, Mak-Soon; Lee, Seohyun; Doo, Miae; Kim, Yangha

    2016-01-01

    Green tea (Camellia sinensis) is one of the most popular beverages in the world and has been acknowledged for centuries as having significant health benefits. (−)-Epigallocatechin-3-gallate (EGCG) is the most abundant catechin in green tea, and it has been reported to have health benefit effects. Peroxisome proliferator-activated receptor γ coactivator (PGC)-1α is a crucial regulator of mitochondrial biogenesis and hepatic gluconeogenesis. The objective of this study was to investigate whether EGCG from green tea can affect the ability of transcriptional regulation on PGC-1α mRNA expression in HepG2 cells and 3T3-L1 adipocytes. To study the molecular mechanism that allows EGCG to control PGC-1α expression, the promoter activity levels of PGC-1α were examined. The PGC-1α mRNA level was measured using quantitative real-time PCR. The −970/+412 bp of PGC-1α promoter was subcloned into the pGL3-Basic vector that includes luciferase as a reporter gene. EGCG was found to up-regulate the PGC-1α mRNA levels significantly with 10 μmol/L of EGCG in HepG2 cells and differentiated 3T3-L1 adipocytes. PGC-1α promoter activity was also increased by treatment with 10 μmol/L of EGCG in both cells. These results suggest that EGCG may induce PGC-1α gene expression, potentially through promoter activation. PMID:27069908

  10. Sirtuin1 promotes osteogenic differentiation through downregulation of peroxisome proliferator-activated receptor γ in MC3T3-E1 cells.

    PubMed

    Qu, Bo; Ma, Yuan; Yan, Ming; Gong, Kai; Liang, Feng; Deng, Shaolin; Jiang, Kai; Ma, Zehui; Pan, Xianming

    2016-09-01

    Osteoporosis is a skeletal disorder characterized by bone loss, resulting in architectural deterioration of the skeleton, decreased bone strength and an increased risk of fragility fractures. Strengthening osteogenesis is an effective way to relieve osteoporosis. Sirtuin1 (Sirt1) is a nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase, which is reported to be involved in improving osteogenesis. Sirt1 targets peroxisome proliferator-activated receptor γ (PPARγ) in the regulation of adipose tissues; however, the molecular mechanism of Sirt1 in osteogenic differentiation is still unknown. PPARγ tends to induce more adipogenic differentiation rather than osteogenic differentiation. Hence, we hypothesized that Sirt1 facilitates osteogenic differentiation through downregulation of PPARγ signaling. Mouse pre-osteoblastic MC3T3-E1 cells were cultured under osteogenic medium. Sirt1 was overexpressed through plasmid transfection. The results showed that high expression of Sirt1 was associated with increased osteogenic differentiation, as indicated by quantitative PCR and Western blot analysis of osteogenic markers, and Von Kossa staining. Sirt1 overexpression also directly and negatively regulated the expression of PPARγ and its downstream molecules. Use of the PPARγ agonist Rosiglitazone, reversed the effects of Sirt1 on osteogenic differentiation. Using constructed luciferase plasmids, we demonstrated a role of Sirt1 in inhibiting PPARγ-induced activity and expression of adipocyte-specific genes, including acetyl-coenzyme A carboxylase (Acc) and fatty acid binding protein 4 (Fabp4). The interaction between Sirt1 and PPARγ was further confirmed using co-immunoprecipitation analysis. Together, these results reveal a novel mechanism for Sirt1 in osteogenic differentiation through downregulation of PPARγ activity. These findings suggest that the Sirt1-PPARγ pathway may represent a potential target for enhancement of osteogenesis and treatment of

  11. Platycodon grandiflorum A. De Candolle Ethanolic Extract Inhibits Adipogenic Regulators in 3T3-L1 Cells and Induces Mitochondrial Biogenesis in Primary Brown Preadipocytes.

    PubMed

    Kim, Hye-Lin; Park, Jinbong; Park, Hyewon; Jung, Yunu; Youn, Dong-Hyun; Kang, JongWook; Jeong, Mi-Young; Um, Jae-Young

    2015-09-01

    This study was designed to evaluate the effects of Platycodon grandiflorum A. DC. ethanolic extract (PG) on obesity in brown/white preadipocytes. The effect of PG on the differentiation and mitochondrial biogenesis of brown adipocytes is still not examined. An in vivo study showed that PG induced weight loss in mice with high-fat-diet-induced obesity. PG successfully suppressed the differentiation of 3T3-L1 cells by down-regulating cellular induction of the peroxisome proliferators activated receptor γ (PPARγ), CCAAT enhancer binding protein α (C/EBPα), lipin-1, and adiponectin but increasing expression of silent mating type information regulation 2 homologue 1 (SIRT1) and the phosphorylation of AMP-activated protein kinase α (AMPKα). The effect of PG on the adipogenic factors was compared with that of its bioactive compound platycodin D. In addition, PG increased expressions of mitochondria-related genes, including uncoupling protein 1 (UCP1), peroxisome proliferator activated receptor-coactivator 1 α (PGC1α), PR domain containing 16 (PRDM16), SIRT3, nuclear respiratory factor (NRF), and cytochrome C (CytC) in primary brown adipocytes. These results indicate that PG stimulates the differentiation of brown adipocytes through modulation of mitochondria-related genes and could offer clinical benefits as a supplement to treat obesity. PMID:26244589

  12. 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. PMID:26049170

  13. 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.

  14. Berberine inhibits SREBP-1-related clozapine and risperidone induced adipogenesis in 3T3-L1 cells.

    PubMed

    Hu, Yueshan; Kutscher, Eric; Davies, Gareth E

    2010-12-01

    Weight gain is a common and potentially serious complication associated with the treatment of second generation antipsychotics such as clozapine and risperidone. Increased peripheral adipogenesis via the SREBP-1 pathway could be one critical mechanism responsible for antipsychotic drug-induced weight gain. Berberine, a botanical alkaloid, has been shown in our previous studies to inhibit adipogenesis in cell and animal models. MTT was used to determine the cytotoxic effects of clozapine and risperidone in combination with berberine. Differentiation of 3T3-L1 cells was monitored by Oil-Red-O staining and the expression of SREBP-1 and related proteins was determined by real-time RT-PCR and western blotting. The results showed that neither clozapine nor risperidone, alone or in combination with berberine had significant effects on cell viability. Eight days treatment with 15 μM clozapine increased adipogenesis by 37.4% and 50 μM risperidone increased adipogenesis by 26.5% during 3T3-L1 cell differentiation accompanied by increased SREBP-1, PPARγ, C/EBPα, LDLR and Adiponectin gene expression. More importantly, the addition of 8 μM berberine diminished the induction of adipogenesis almost completely accompanied by down-regulated mRNA and protein expression levels of SREBP-1-related proteins. These encouraging results may lead to the use of berberine as an adjuvant to prevent weight gain during second generation antipsychotic medication. PMID:20564506

  15. Ultrastructural evidence for the accumulation of insulin in nuclei of intact 3T3-L1 adipocytes by an insulin-receptor mediated process

    SciTech Connect

    Smith, R.M.; Jarett, L.

    1987-01-01

    Monomeric ferritin-labeled insulin (F/sub m/-Ins), a biologically active, electron-dense marker of occupied insulin receptors, was used to characterize the internalization of insulin in 3T3-L1 adipocytes. F/sub m/-Ins bound specifically to insulin receptors and was internalized in a time- and temperature-dependent manner. In the nucleus, several F/sub m/-Ins particles usually were found in the same general location-near nuclear pores, associated with the periphery of the condensed chromatin. Addition of a 250-fold excess of unlabeled insulin or incubation at 15/sup 0/C reduced the number of F/sub m/-Ins particles found in nuclei after 90 min by 99% or 92%, respectively. Nuclear accumulation of unlabeled ferritin was only 2% of that found with F/sub m/-Ins after 90 min at 37/sup 0/C. Biochemical experiments utilizing /sup 125/I-labeled insulin and subcellular fractionation indicated that intact 3T3-L1 adipocytes internalized insulin rapidly and that approx. = 3% of the internalized ligand accumulated in nuclei after 1 hr. These data provide biochemical and high-resolution ultrastructural evidence that 3T3-L1 adipocytes accumulate potentially significant amounts of insulin in nuclei by an insulin receptor-mediated process. The transport of insulin or the insulin-receptor complex to nuclei in this cell or in others may be directly involved in the long-term biological effects of insulin - in particular, in the control of DNA and RNA synthesis.

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

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

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

    NASA Technical Reports Server (NTRS)

    Fitzgerald, J.; Hughes-Fulford, M.

    1999-01-01

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

  18. Polymethoxyflavonoids from Kaempferia parviflora induce adipogenesis on 3T3-L1 preadipocytes by regulating transcription factors at an early stage of differentiation.

    PubMed

    Horikawa, Takumi; Shimada, Tsutomu; Okabe, Yui; Kinoshita, Kaoru; Koyama, Kiyotaka; Miyamoto, Ken-ichi; Ichinose, Koji; Takahashi, Kunio; Aburada, Masaki

    2012-01-01

    We previously reported that Kaempferia parviflora WALL. ex BAKER (KP) and its ethyl acetate extract (KPE) improve various metabolic disorders in obesity-model mice. However the mechanism is not certain, and, in this study, in order to elucidate the mechanism of the suppressive effect of KP on fat accumulation, we focused on adipocytes, which are closely linked to metabolic diseases. The finding was that KPE and its components, 3,5,7,4'-tetramethoxyflavone and 3,5,7,3',4'-pentamethoxyflavone, strongly induced differentiation of 3T3-L1 preadipocytes to adipocytes. The above two polymethoxyflavonoids (PMFs) also induced adiponectin mRNA levels, and release of adiponectin into the medium. In addition, these PMFs enhanced the expression of peroxisome proliferator-activated receptor γ (PPARγ), but did not show PPARγ ligand activity. We then investigated the expression of the differentiation-regulator located upstream of PPARγ. Expression of CCAAT/enhancer-binding protein (C/EBP) β and -δ mRNA, a transcriptional regulator of PPARγ, was induced, and expression of GATA-2 mRNA, a down-regulator of adipogenesis, was suppressed by these PMFs. These functions of the KP PMFs that enhance adipogenesis and secretion of adiponectin are, to some extent at least, involved in the mechanisms of anti-metabolic disorders effects. PMID:22687402

  19. E2a-Pbx1 induces aberrant expression of tissue-specific and developmentally regulated genes when expressed in NIH 3T3 fibroblasts.

    PubMed Central

    Fu, X; Kamps, M P

    1997-01-01

    The E2a-Pbx1 oncoprotein contains the transactivation domain of E2a joined to the DNA-binding homeodomain (HD) of Pbx1. In mice, E2a-Pbx1 transforms T lymphoblasts and fibroblasts and blocks myeloblast differentiation. Pbx1 and E2a-Pbx1 bind DNA as heterodimers with other HD proteins whose expression is tissue specific. While the transactivation domain of E2a is required for all forms of transformation, DNA binding by the Pbx1 HD is essential for blocking myeloblast differentiation but dispensable for fibroblast or T-lymphoblast transformation. These properties suggest (i) that E2a-Pbx1 causes cellular transformation by activating gene transcription, (ii) that transcription of E2a-Pbx1 target genes is normally regulated by ubiquitous Pbx proteins and tissue-specific partners, and (iii) that DNA-binding mutants of E2a-Pbx1 activate a subset of all gene targets. To test these predictions, genes induced in NIH 3T3 fibroblasts by E2a-Pbx1 were identified and examined for tissue- and stage-specific expression and their differential abilities to be upregulated by E2a-Pbx1 in NIH 3T3 fibroblasts and myeloblasts and by a DNA-binding mutant of E2a-Pbx1 in NIH 3T3 cells. Of 12 RNAs induced by E2a-Pbx1, 4 encoded known proteins (a J-C region of the immunoglobulin kappa light chain, natriuretic peptide receptor C, mitochondrial fumarase, and the 3',5'-cyclic nucleotide phosphodiesterase, PDE1A) and 5 encoded new proteins related to angiogenin, ion channels, villin, epidermal growth factor repeat proteins, and the human 2.19 gene product. Expression of many of these genes was tissue specific or developmentally regulated, and most were not expressed in fibroblasts, indicating that E2a-Pbx1 can induce ectopic expression of genes associated with lineage-specific differentiation. PMID:9032278

  20. DMSO is a strong inducer of DNA hydroxymethylation in pre-osteoblastic MC3T3-E1 cells

    PubMed Central

    Thaler, Roman; Spitzer, Silvia; Karlic, Heidrun; Klaushofer, Klaus; Varga, Franz

    2012-01-01

    Artificial induction of active DNA demethylation appears to be a possible and useful strategy in molecular biology research and therapy development. Dimethyl sulfoxide (DMSO) was shown to cause phenotypic changes in embryonic stem cells altering the genome-wide DNA methylation profiles. Here we report that DMSO increases global and gene-specific DNA hydroxymethylation levels in pre-osteoblastic MC3T3-E1 cells. After 1 day, DMSO increased the expression of genes involved in DNA hydroxymethylation (TET) and nucleotide excision repair (GADD45) and decreased the expression of genes related to DNA methylation (Dnmt1, Dnmt3b, Hells). Already 12 hours after seeding, before first replication, DMSO increased the expression of the pro-apoptotic gene Fas and of the early osteoblastic factor Dlx5, which proved to be Tet1 dependent. At this time an increase of 5-methyl-cytosine hydroxylation (5-hmC) with a concomitant loss of methyl-cytosines on Fas and Dlx5 promoters as well as an increase in global 5-hmC and loss in global DNA methylation was observed. Time course-staining of nuclei suggested euchromatic localization of DMSO induced 5-hmC. As consequence of induced Fas expression, caspase 3/7 and 8 activities were increased indicating apoptosis. After 5 days, the effect of DMSO on promoter- and global methylation as well as on gene expression of Fas and Dlx5 and on caspases activities was reduced or reversed indicating down-regulation of apoptosis. At this time, up regulation of genes important for matrix synthesis suggests that DMSO via hydroxymethylation of the Fas promoter initially stimulates apoptosis in a subpopulation of the heterogeneous MC3T3-E1 cell line, leaving a cell population of extra-cellular matrix producing osteoblasts.  PMID:22507896

  1. 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

  2. Angiotensin II Requires Zinc and Downregulation of the Zinc Transporters ZnT3 and ZnT10 to Induce Senescence of Vascular Smooth Muscle Cells

    PubMed Central

    Patrushev, Nikolay; Seidel-Rogol, Bonnie; Salazar, Gloria

    2012-01-01

    Senescence, a hallmark of mammalian aging, is associated with the onset and progression of cardiovascular disease. Angiotensin II (Ang II) signaling and zinc homeostasis dysfunction are increased with age and are linked to cardiovascular disease, but the relationship among these processes has not been investigated. We used a model of cellular senescence induced by Ang II in vascular smooth muscle cells (VSMCs) to explore the role of zinc in vascular dysfunction. We found that Ang II-induced senescence is a zinc-dependent pathway mediated by the downregulation of the zinc transporters ZnT3 and ZnT10, which work to reduce cytosolic zinc. Zinc mimics Ang II by increasing reactive oxygen species (ROS), activating NADPH oxidase activity and Akt, and by downregulating ZnT3 and ZnT10 and inducing senescence. Zinc increases Ang II-induced senescence, while the zinc chelator TPEN, as well as overexpression of ZnT3 or ZnT10, decreases ROS and prevents senescence. Using HEK293 cells, we found that ZnT10 localizes in recycling endosomes and transports zinc into vesicles to prevent zinc toxicity. Zinc and ZnT3/ZnT10 downregulation induces senescence by decreasing the expression of catalase. Consistently, ZnT3 and ZnT10 downregulation by siRNA increases ROS while downregulation of catalase by siRNA induces senescence. Zinc, siZnT3 and siZnT10 downregulate catalase by a post-transcriptional mechanism mediated by decreased phosphorylation of ERK1/2. These data demonstrate that zinc homeostasis dysfunction by decreased expression of ZnT3 or ZnT10 promotes senescence and that Ang II-induced senescence is a zinc and ROS-dependent process. Our studies suggest that zinc might also affect other ROS-dependent processes induced by Ang II, such as hypertrophy and migration of smooth muscle cells. PMID:22427991

  3. Trans, trans-farnesol as a mevalonate-derived inducer of murine 3T3-F442A pre-adipocyte differentiation.

    PubMed

    Torabi, Sheida; Mo, Huanbiao

    2016-03-01

    Based on our finding that depletion of mevalonate-derived metabolites inhibits adipocyte differentiation, we hypothesize that trans, trans-farnesol (farnesol), a mevalonate-derived sesquiterpene, induces adipocyte differentiation. Farnesol dose-dependently (25-75 μmol/L) increased intracellular triglyceride content of murine 3T3-F442A pre-adipocytes measured by AdipoRed™ Assay and Oil Red-O staining. Concomitantly, farnesol dose-dependently increased glucose uptake and glucose transport protein 4 (GLUT4) expression without affecting cell viability. Furthermore, quantitative real-time polymerase chain reaction and Western blot showed that farnesol increased the mRNA and protein levels of peroxisome proliferator-activated receptor γ (PPARγ), a key regulator of adipocyte differentiation, and the mRNA levels of PPARγ-regulated fatty acid-binding protein 4 and adiponectin; in contrast, farnesol downregulated Pref-1 gene, a marker of pre-adipocytes. GW9662 (10 µmol/L), an antagonist of PPARγ, reversed the effects of farnesol on cellular lipid content, suggesting that PPARγ signaling pathway may mediate the farnesol effect. Farnesol (25-75 μmol/L) did not affect the mRNA level of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in the mevalonate pathway. Farnesol may be the mevalonate-derived inducer of adipocyte differentiation and potentially an insulin sensitizer via activation of PPARγ and upregulation of glucose uptake. PMID:26660152

  4. The effects of propionate and valerate on insulin responsiveness for glucose uptake in 3T3-L1 adipocytes and C2C12 myotubes via G protein-coupled receptor 41.

    PubMed

    Han, Joo-Hui; Kim, In-Su; Jung, Sang-Hyuk; Lee, Sang-Gil; Son, Hwa-Young; Myung, Chang-Seon

    2014-01-01

    Since insulin resistance can lead to hyperglycemia, improving glucose uptake into target tissues is critical for regulating blood glucose levels. Among the free fatty acid receptor (FFAR) family of G protein-coupled receptors, GPR41 is known to be the Gαi/o-coupled receptor for short-chain fatty acids (SCFAs) such as propionic acid (C3) and valeric acid (C5). This study aimed to investigate the role of GPR41 in modulating basal and insulin-stimulated glucose uptake in insulin-sensitive cells including adipocytes and skeletal muscle cells. Expression of GPR41 mRNA and protein was increased with maximal expression at differentiation day 8 for 3T3-L1 adipocytes and day 6 for C2C12 myotubes. GPR41 protein was also expressed in adipose tissues and skeletal muscle. After analyzing dose-response relationship, 300 µM propionic acid or 500 µM valeric acid for 30 min incubation was used for the measurement of glucose uptake. Both propionic acid and valeric acid increased insulin-stimulated glucose uptake in 3T3-L1 adipocyte, which did not occur in cells transfected with siRNA for GPR41 (siGPR41). In C2C12 myotubes, these SCFAs increased basal glucose uptake, but did not potentiate insulin-stimulated glucose uptake, and siGPR41 treatment reduced valerate-stimulated basal glucose uptake. Therefore, these findings indicate that GPR41 plays a role in insulin responsiveness enhanced by both propionic and valeric acids on glucose uptake in 3T3-L1 adipocytes and C2C12 myotubes, and in valerate-induced increase in basal glucose uptake in C2C12 myotubes. PMID:24748202

  5. Curcumin improves hypoxia induced dysfunctions in 3T3-L1 adipocytes by protecting mitochondria and down regulating inflammation.

    PubMed

    Priyanka, Ariyapalli; Anusree, Sasidharan Suseela; Nisha, Vijayakumar Marykutty; Raghu, Kozhiparambil Gopalan

    2014-01-01

    Obesity induced metabolic syndrome is increasing worldwide at an alarming rate. It is characterized by excessive expansion of white adipose tissue which leads to hypoxia and impairs normal metabolism. Recent studies reveal that hypoxia could be one of the factors for inflammation, insulin resistance and other obesity related complications. There is a high demand for anti-obese phytoceuticals to control and manage the complications resulting from obesity. In this study, we investigated how hypoxia affect the physiological functions of 3T3-L1 adipocytes emphasizing on oxidative stress, inflammation, and mitochondrial functions. We also evaluated the protective role of various doses of curcumin, a well-known dietary antioxidant, on hypoxia induced alterations. The results revealed that hypoxia significantly altered the vital parameters of adipocyte biology like HIF 1α expression (103.47% ↑), lactate, and glycerol release (184.34% and 69.1% ↑, respectively), reactive oxygen species production (432.53% ↑), lipid and protein oxidation (376.6% and 566.6% ↑, respectively), reduction in antioxidant enzymes (superoxide dismutase and catalase) status, secretion of inflammatory markers (TNF α, IL 6, IL 1β, and IFN γ), and mitochondrial functions (mitochondrial mass, membrane potential, permeability transition pore integrity, and superoxide generation). Curcumin substantially protected adipocytes from toxic effects of hypoxia in a dose dependent manner by protecting mitochondria and down regulating inflammation. Acriflavine is used as a positive control. A detailed investigation is required for the development of curcumin as an effective nutraceutical against obesity. PMID:25110893

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2010-09-01

    Dietary conjugated linoleic acid (CLA) has been reported to exhibit a number of therapeutic effects in animal models and patients, such as anti-hypertensive, anti-hyperlipidemic, anti-arteriosclerotic, anti-carcinogenic, and anti-diabetic effects. However, the underlying mechanism is not well-characterized. In the present study, the effects of cis(c)9, trans(t)11-CLA on the differentiation of mouse 3T3-L1 preadipocytes into mature adipocytes were examined. Treatment with c9, t11-CLA in the presence of insulin, dexamethasone, and 3-isobutyl-1-methyl-xanthine (differentiation cocktail) significantly stimulated the accumulation of triacylglycerol. The microscopic observation of cells stained by Oil Red O demonstrated that c9, t11-CLA increases the amount and proportion of small mature adipocytes secreting adiponectin, a benign adipocytokine, when compared to the differentiation cocktail alone. Furthermore, c9, t11-CLA increased bioactive peroxisome proliferator-activated receptor γ (PPARγ) levels in a nuclear extract of 3T3-L1 cells, suggesting the enhancing effect of this fatty acid on the nuclear transmission of PPARγ, a master regulator of adipocyte differentiation, in 3T3-L1 cells. These results suggest that the therapeutic effects of c9, t11-CLA on lifestyle-related diseases are partially due to the enhanced formation of small adipocytes from preadipocytes via PPARγ stimulation. PMID:20838573

  8. 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.

  9. 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. PMID:22732655

  10. Zinc transporter 3 (ZnT3) gene deletion reduces spinal cord white matter damage and motor deficits in a murine MOG-induced multiple sclerosis model.

    PubMed

    Choi, Bo Young; Kim, In Yeol; Kim, Jin Hee; Kho, A Ra; Lee, Song Hee; Lee, Bo Eun; Sohn, Min; Koh, Jae-Young; Suh, Sang Won

    2016-10-01

    The present study aimed to evaluate the role of zinc transporter 3 (ZnT3) on multiple sclerosis (MS) pathogenesis. Experimental autoimmune encephalomyelitis (EAE), a disease model of multiple sclerosis, was induced by immunization with myelin oligodendrocyte glycoprotein (MOG35-55) in female mice. Three weeks after the initial immunization, demyelination, immune cell infiltration and blood brain barrier (BBB) disruption in the spinal cord were analyzed. Clinical signs of EAE first appeared on day 11 and reached a peak level on day 19 after the initial immunization. ZnT3 gene deletion profoundly reduced the daily clinical score of EAE. The ZnT3 gene deletion-mediated inhibition of the clinical course of EAE was accompanied by suppression of inflammation and demyelination in the spinal cord. The motor deficit accompanying neuropathological changes associated with EAE were mild in ZnT3 gene deletion mice. This reduction in motor deficit was accompanied by coincident reductions in demyelination and infiltration of encephalitogenic immune cells including CD4+ T cells, CD8+ T cells, CD20+ B cells and F4/80+ microglia in the spinal cord. These results demonstrate that ZnT3 gene deletion inhibits the clinical features and neuropathological changes associated with EAE. ZnT3 gene deletion also remarkably inhibited formation of EAE-associated aberrant synaptic zinc patches, matrix metalloproteinases-9 (MMP-9) activation and BBB disruption. Therefore, amelioration of EAE-induced clinical and neuropathological changes by ZnT3 gene deletion suggests that vesicular zinc may be involved in several steps of MS pathogenesis. PMID:27370228

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

    SciTech Connect

    Murayama, T.; Ui, M.

    1985-06-25

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

  12. IL-17A synergistically enhances TNFα-induced IL-6 and CCL20 production in 3T3-L1 adipocytes.

    PubMed

    Shinjo, Takanori; Iwashita, Misaki; Yamashita, Akiko; Sano, Tomomi; Tsuruta, Mitsudai; Matsunaga, Hiroaki; Sanui, Terukazu; Asano, Tomoichiro; Nishimura, Fusanori

    2016-08-19

    Interleukin-17A (IL-17A) is known to induce inflammatory responses and to be involved in the pathogenesis of not only autoimmune diseases, but also several metabolic and infectious diseases. In this study, IL-17A is shown to induce IL-6 expression in 3T3-L1 mature adipocytes. Interestingly, we found that IL-17A synergistically amplified TNFα-induced secretion of IL-6 and upregulation of IL-17RA expression in 3T3-L1 adipocytes. Its synergistic effects on IL-6 production were inhibited by pre-treatment with inhibitors of IκBα and JNK. Furthermore, IL-17A cooperatively enhanced LPS-mediated IL-6 production in 3T3-L1 adipocytes co-cultured with RAW264.7 macrophages. In addition, IL-17A also enhanced CCL20 production in 3T3-L1 adipocytes stimulated with TNFα or co-cultured with LPS-stimulated RAW macrophages. In high-fat diet-fed mouse epididymal adipose tissues, IL-17RA and RORγt mRNA levels were significantly increased and the serum level of CCL20 was also upregulated. Taken together, these data show that, in adipose tissues, IL-17A contributes to exacerbating insulin resistance-enhancing IL-6 production and promotes the infiltration of Th17 cells in cooperation with TNFα; these findings represent a novel hypothesis for the association between IL-17A-producing cells and type 2 diabetes. PMID:27311858

  13. Functional binding of a vertebrate hormone, L-3,5,3'-triiodothyronine (T3), on insect follicle cell membranes.

    PubMed

    Kim, Y; Davari, E D; Sevala, V; Davey, K G

    1999-10-01

    A vertebrate hormone, L-3,5,3'-triiodothyronine (T3), induces volume reduction in the follicle cells of Locusta migratoria and Rhodnius prolixus. The effect of T3 on locust follicle cells is inhibited by ouabain and by antibodies raised against a membrane binding protein for juvenile hormone (JH). [125I]-T3 binds to membrane preparations of vitellogenic follicles in a specific and saturable fashion, with a KD in the low nanomolar range. T3 and JH III exhibited equivalent abilities to compete for the T3 binding site. These findings strongly suggest that T3 and JH act via the same receptor in follicle cells. PMID:10528413

  14. Ghrelin protects against depleted uranium-induced apoptosis of MC3T3-E1 cells through oxidative stress-mediated p38-mitogen-activated protein kinase pathway.

    PubMed

    Hao, Yuhui; Liu, Cong; Huang, Jiawei; Gu, Ying; Li, Hong; Yang, Zhangyou; Liu, Jing; Wang, Weidong; Li, Rong

    2016-01-01

    Depleted uranium (DU) mainly accumulates in the bone over the long term. Osteoblast cells are responsible for the formation of bone, and they are sensitive to DU damage. However, studies investigating methods of reducing DU damage in osteoblasts are rarely reported. Ghrelin is a stomach hormone that stimulates growth hormones released from the hypothalamic-pituitary axis, and it is believed to play an important physiological role in bone metabolism. This study evaluates the impact of ghrelin on DU-induced apoptosis of the osteoblast MC3T3-E1 and investigates its underlying mechanisms. The results show that ghrelin relieved the intracellular oxidative stress induced by DU, eliminated reactive oxygen species (ROS) and reduced lipid peroxidation by increasing intracellular GSH levels; in addition, ghrelin effectively suppressed apoptosis, enhanced mitochondrial membrane potential, and inhibited cytochrome c release and caspase-3 activation after DU exposure. Moreover, ghrelin significantly reduced the expression of DU-induced phosphorylated p38-mitogen-activated protein kinase (MAPK). A specific inhibitor (SB203580) or specific siRNA of p38-MAPK could significantly suppress DU-induced apoptosis and related signals, whereas ROS production was not affected. In addition, ghrelin receptor inhibition could reduce the anti-apoptosis effect of ghrelin on DU and reverse the effect of ghrelin on intracellular ROS and p38-MAPK after DU exposure. These results suggest that ghrelin can suppress DU-induced apoptosis of MC3T3-E1 cells, reduce DU-induced oxidative stress by interacting with its receptor, and inhibit downstream p38-MAPK activation, thereby suppressing the mitochondrial-dependent apoptosis pathway. PMID:26529667

  15. T3 (Triiodothyronine) Test

    MedlinePlus

    ... be limited. Home Visit Global Sites Search Help? Free T3 and Total T3 Share this page: Was ... helpful? Also known as: FT3; Triiodothyronine Formal name: Free Triiodothyronine; Total Triiodothyronine Related tests: TSH ; Free T4 ; ...

  16. T3 test

    MedlinePlus

    ... thyroid function. For example, in some cases of hyperthyroidism , T3 may be increased but T4 may be ... More Chronic Chronic thyroiditis (Hashimoto disease) Graves disease Hyperthyroidism Hypothyroidism Protein in diet Silent thyroiditis T3RU test ...

  17. 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. PMID:26605160

  18. 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

  19. NOD1 activation induces proinflammatory gene expression and insulin resistance in 3T3-L1 adipocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chronic inflammation is associated with obesity and insulin resistance. However, the underlying mechanisms are not fully understood. Pattern recognition receptors Toll-like receptors and Nucleotide-oligomerization domain containing proteins play critical roles in innate immune response. Here we repo...

  20. T3RU test

    MedlinePlus

    Resin T3 uptake; T3 resin uptake; Thyroid hormone-binding ratio ... This test is done to check your thyroid function. Thyroid function depends on the action of many different hormones, including thyroid-stimulating hormone (TSH), T3, and T4. This test helps see ...

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

    PubMed

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

    2014-12-01

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

  2. Expression of Estrogen Receptor Beta Predicts Oncologic Outcome of pT3 Upper Urinary Tract Urothelial Carcinoma Better Than Aggressive Pathological Features

    PubMed Central

    Luo, Hao Lun; Sung, Ming Tse; Tsai, Eing Mei; Lin, Chang Shen; Lee, Nai Lun; Chung, Yueh-Hua; Chiang, Po Hui

    2016-01-01

    Upper urinary tract urothelial carcinoma (UT-UC) is rare and treatment options or prognostic markers are limited. There is increasing evidence indicating that urothelial carcinoma may be an endocrine-related cancer. The aim of this study was to analyze the prognostic effect of estrogen receptor beta (ERβ) on the outcome of UT-UC. From 2005 to 2012, this study included 105 patients with pT3 UT-UC. Perioperative factors, pathological features, and ERβ immunostaining were reviewed and prognostic effects were examined by multivariate analysis. This study divided patients into either the ERβ-high (n = 52) or ERβ-low (n = 53) group and analyzed their oncologic outcomes. All pathological features except infiltrating tumor architecture (significantly higher incidence in ERβ-low group, p = 0.004) are symmetric in both groups. Low ERβ expression was significantly correlated with local recurrence and distant metastasis in univariate analysis (p = 0.035 and 0.004, respectively) and multivariate analysis (p = 0.05 and 0.008, respectively). Cell line study also proved that knock down of ERβ cause less UTUC proliferation and migration. In addition, ERβ agonist also enhanced the cytotoxic and migration inhibition effect of cisplatin and ERβ antagonist cause the UTUC cell more resistant to cisplatin. This result may help identify patients in need of adjuvant therapy or develop potential targeted therapy. PMID:27052470

  3. 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.

  4. 12/15-lipoxygenase products induce inflammation and impair insulin signaling in 3T3-L1 adipocytes.

    PubMed

    Chakrabarti, Swarup K; Cole, Banumathi K; Wen, Yeshao; Keller, Susanna R; Nadler, Jerry L

    2009-09-01

    Inflammation and insulin resistance associated with visceral obesity are important risk factors for the development of type 2 diabetes, atherosclerosis, and the metabolic syndrome. The 12/15-lipoxygenase (12/15-LO) enzyme has been linked to inflammatory changes in blood vessels that precede the development of atherosclerosis. The expression and role of 12/15-LO in adipocytes have not been evaluated. We found that 12/15-LO mRNA was dramatically upregulated in white epididymal adipocytes of high-fat fed mice. 12/15-LO was poorly expressed in 3T3-L1 fibroblasts and was upregulated during differentiation into adipocytes. Interestingly, the saturated fatty acid palmitate, a major component of high fat diets, augmented expression of 12/15-LO in vitro. When 3T3-L1 adipocytes were treated with the 12/15-LO products, 12-hydroxyeicosatetranoic acid (12(S)-HETE) and 12-hydroperoxyeicosatetraenoic acid (12(S)-HPETE), expression of proinflammatory cytokine genes, including tumor necrosis factor-alpha (TNF-alpha), monocyte chemoattractant protein 1 (MCP-1), interleukin 6 (IL-6), and IL-12p40, was upregulated whereas anti-inflammatory adiponectin gene expression was downregulated. 12/15-LO products also augmented c-Jun N-terminal kinase 1 (JNK-1) phosphorylation, a known negative regulator of insulin signaling. Consistent with impaired insulin signaling, we found that insulin-stimulated 3T3-L1 adipocytes exhibited decreased IRS-1(Tyr) phosphorylation, increased IRS-1(Ser) phosphorylation, and impaired Akt phosphorylation when treated with 12/15-LO product. Taken together, our data suggest that 12/15-LO products create a proinflammatory state and impair insulin signaling in 3T3-L1 adipocytes. Because 12/15-LO expression is upregulated in visceral adipocytes by high-fat feeding in vivo and also by addition of palmitic acid in vitro, we propose that 12/15-LO plays a role in promoting inflammation and insulin resistance associated with obesity. PMID:19521344

  5. 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

  6. Mammalian target of rapamycin complex 1 (mTORC1) plays a role in Pasteurella multocida toxin (PMT)-induced protein synthesis and proliferation in Swiss 3T3 cells.

    PubMed

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

    2013-01-25

    Pasteurella multocida toxin (PMT) is a potent mitogen known to activate several signaling pathways via deamidation of a conserved glutamine residue in the α subunit of heterotrimeric G-proteins. However, the detailed mechanism behind mitogenic properties of PMT is unknown. Herein, we show that PMT induces protein synthesis, cell migration, and proliferation in serum-starved Swiss 3T3 cells. Concomitantly PMT induces phosphorylation of ribosomal S6 kinase (S6K1) and its substrate, ribosomal S6 protein (rpS6), in quiescent 3T3 cells. The extent of the phosphorylation is time and PMT concentration dependent, and is inhibited by rapamycin and Torin1, the two specific inhibitors of the mammalian target of rapamycin complex 1 (mTORC1). Interestingly, PMT-mediated mTOR signaling activation was observed in MEF WT but not in Gα(q/11) knock-out cells. These observations are consistent with the data indicating that PMT-induced mTORC1 activation proceeds via the deamidation of Gα(q/11), which leads to the activation of PLCβ to generate diacylglycerol and inositol trisphosphate, two known activators of the PKC pathway. Exogenously added diacylglycerol or phorbol 12-myristate 13-acetate, known activators of PKC, leads to rpS6 phosphorylation in a rapamycin-dependent manner. Furthermore, PMT-induced rpS6 phosphorylation is inhibited by PKC inhibitor, Gö6976. Although PMT induces epidermal growth factor receptor activation, it exerts no effect on PMT-induced rpS6 phosphorylation. Together, our findings reveal for the first time that PMT activates mTORC1 through the Gα(q/11)/PLCβ/PKC pathway. The fact that PMT-induced protein synthesis and cell migration is partially inhibited by rapamycin indicates that these processes are in part mediated by the mTORC1 pathway. PMID:23223576

  7. IL-1α induces apoptosis and inhibits the osteoblast differentiation of MC3T3-E1 cells through the JNK and p38 MAPK pathways.

    PubMed

    Guo, Chun; Yang, Xu-Guang; Wang, Fei; Ma, Xu-Yuan

    2016-07-01

    Interleukin (IL)-1 is a proinflammatory cytokine that plays important roles in inflammation and host responses to infection. The present study aimed to evaluate the effects of IL-1α on the apoptosis and differentiation of osteoblasts, and to elucidate the mechanism responsible for these effects in the osteoblast‑like cell line MC3T3-E1. The MC3T3-E1 cells were non-treated or treated with IL-1α. Following treatment, cell viability, alkaline phosphatase (ALP) activity and caspase-3 activity were evaluated. The expression of osteoblast-specific genes as well as Bax, Bcl-2 and caspase-3 were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The protein levels of Bax, Bcl-2, caspase-3 and the phosphorylation of mitogen-activated protein kinases (MAPKs, also known as MAP kinases) were evaluated using western blot analysis. The MAPK signaling pathway was blocked by pre-treatment with MAPK inhibitors SB203580, PD98059 and SP600125. IL-1α treatment induced a significant decrease in cell viability and ALP activity in the MC3T3-E1 cells. IL-1α also significantly decreased the mRNA expression and protein levels of osteoblast-related genes in the MC3T3-E1 cells. On the other hand, IL-1α significantly upregulated the mRNA expression and protein levels of Bax and caspase-3 as well as caspase-3 activity, whereas Bcl-2 expression was decreased in the MC3T3-E1 cells. Furthermore, IL-1α activated the apoptotic signaling pathway by increasing the phosphorylation of c-Jun N-terminal kinase (JNK) and p38-MAPK, whereas it inhibited the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). Moreover, pre-treatment with MAPK inhibitors attenuated the phosphorylation of JNK, p38 and Bax expression enhanced by IL-1α. However, MAPK inhibitors markedly increased the protein expression of osteoblast-related genes and Bcl-xL in the MC3T3-E1 cells downregulated by IL-1α. Taken together, these findings suggest that IL-1α induces

  8. Effect of fluoride on insulin level of rats and insulin receptor expression in the MC3T3-E1 cells.

    PubMed

    Hu, Chun-Yan; Ren, Li-Qun; Li, Xi-Ning; Wu, Nan; Li, Guang-Sheng; Liu, Qin-Yi; Xu, Hui

    2012-12-01

    Studies on the role of insulin and insulin receptor (InsR) in the process of skeletal fluorosis, especially in osteogenic function, are rare. We evaluated the effect of increasing F⁻ doses on the marker of bone formation, serum insulin level and pancreatic secretion changes in vivo and mRNA expression of InsR and osteocalcin (OCN) in vitro. Wistar rats (n = 50) were divided into two groups, i.e. a control group and fluoride group. The fluoride groups were treated with fluoride by drinking tap water containing 100 mg F⁻/L. The fluoride ion-selective electrode measured the fluoride concentrations of femurs. The alkaline phosphatase (ALP), OCN, insulin and glucagon of serum were tested to observe the effect of fluoride action on them. Meantime, the pancreas pathological morphometry analysis via β cells stained by aldehyde fuchsin showed the action of fluoride on pancreas secretion. MC3T3-E1 cells (derived from newborn mouse calvaria) were exposed to varying concentrations and periods of fluoride. The mRNA expression of InsR and OCN was quantified with real-time PCR. Results showed that 1-year fluoride treatment obviously stimulated ALP activity and OCN level along with increase of bone fluoride concentration of rats, which indicated that fluoride obviously stimulated osteogenic action of rats. In vitro study, the dual effect of fluoride on osteoblast function is shown. On the other hand, there was a significant increase of serum insulin level and a general decrease of glucagon level, and the histomorphometry analysis indicated an elevated insulin-positive area and increase in islet size in rats treated with fluoride for 1 year. In addition, fluoride obviously facilitated the mRNA expression of InsR in vitro. To sum up, there existed a close relationship between insulin secretion and fluoride treatment. The insulin signal pathway might be involved in the underlying occurrence or development of skeletal fluorosis. PMID:22872571

  9. Novel ATP-binding heat-inducible protein of Mr = 37,000 that is sensitive to transformation in BALB/3T3 cells

    SciTech Connect

    Nakai, A.; Hirayama, C.; Ohtsuka, K.; Hirayoshi, K.; Nagata, K. )

    1990-06-01

    Using affinity chromatography on ATP-agarose, we have identified a major ATP-binding protein in Nonidet P-40 extracts of avian and mammalian cells labeled with (35S)methionine. After washing ATP-agarose beads with high-ionic-strength buffer (0.4 M NaCl), the 37-kD protein was shown to be one of the major ATP-binding proteins while p72 and grp78, which are members of the hsp70 family, also bound to ATP-agarose. This protein consisted of several spots on two-dimensional gel electrophoresis. The isoelectric point of the most basic spot was approximately 9.2 in chick embryo fibroblasts, whereas it was about 8.8 in mouse 3T3 cells. The identities of these proteins in mouse and chick cells were confirmed by peptide mapping. After heat-shock treatment of BALB/3T3 cells, the major heat-shock protein, hsp70, was shown to be induced very rapidly after heat shock and was recovered in the ATP-binding fraction. Besides hsp70, a 37-kD protein was also found to be induced by heat shock. This protein was drastically induced by treating the cells with alpha,alpha'-dipyridyl, an iron chelating reagent, but not with sodium arsenite, calcium ionophore, or tunicamycin. The synthesis and the total amount of this ATP-binding protein increased in mouse 3T3 cells transformed by simian virus 40, methylcholanthrene, or activated c-Ha-ras oncogene compared to their normal counterparts. The incorporation of (32P)orthophosphate was not detected in either normal or transformed cells. These studies established that a major ATP-binding protein of Mr = 37,000 is a heat-inducible protein and that the synthesis of this protein is regulated by malignant transformation.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2015-01-01

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

  12. Methionine restriction inhibits chemically-induced malignant transformation in the BALB/c 3T3 cell transformation assay.

    PubMed

    Nicken, Petra; Empl, Michael T; Gerhard, Daniel; Hausmann, Julia; Steinberg, Pablo

    2016-09-01

    High consumption of red meat entails a higher risk of developing colorectal cancer. Methionine, which is more frequently a component of animal proteins, and folic acid are members of the one carbon cycle and as such important players in DNA methylation and cancer development. Therefore, dietary modifications involving altered methionine and folic acid content might inhibit colon cancer development. In the present study, the BALB/c 3T3 cell transformation assay was used to investigate whether methionine and folic acid are able to influence the malignant transformation of mouse fibroblasts after treatment with the known tumour initiator 3-methylcholanthrene. Three different methionine concentrations (representing a -40%, a "normal" and a +40% cell culture medium concentration, respectively) and two different folic acid concentrations (6 and 20 μM) were thereby investigated. Methionine restriction led to a decrease of type III foci, while enhancement of both methionine and folic acid did not significantly increase the cell transformation rate. Interestingly, the focus-lowering effect of methionine was only significant in conjunction with an elevated folic acid concentration. In summary, we conclude that the malignant transformation of mouse fibroblasts is influenced by methionine levels and that methionine restriction could be a possible approach to reduce cancer development. PMID:27427305

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    Singh, Jyotsna; Kakkar, Poonam

    2014-10-15

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

  15. Ionomycin induces prostaglandin E2 formation in murine osteoblastic MC3T3-E1 cells via mechanisms independent of its ionophoric nature.

    PubMed

    Leis, Hans Jörg; Windischhofer, Werner

    2016-06-01

    Ionomycin and A23187 are divalent cation ionophores with a marked preference for calcium. Studies using these ionophores have almost exclusively interpreted their results in the light of calcium elevation. It was the aim of this study to investigate the effects of ionomycin in osteoblatic MC3T3-E1 cells that are not attributable to its ionophoric properties. Thus, we have found that in contrast to A23187, ionomycin shows similar effects on prostaglandin E2 formation as bradykinin and endothelin-1, being potentiated by extracellular nickel and inhibited by cholera toxin and pertussis toxin. Our data strongly suggest that inomycin, at least in part, exerts its effects via specific binding to a G-protein coupled receptor, thereby evoking downstream cellular events like arachidonate release with subsequent prostaglandin formation. PMID:27065246

  16. T3RU test

    MedlinePlus

    ... sample is needed. How to Prepare for the Test Your health care provider will tell you if you need to stop taking medicines that may affect the test result. Drugs that can increase T3RU levels include: ...

  17. Expression of Caveolin-1 reduces cellular responses to TGF-{beta}1 through down-regulating the expression of TGF-{beta} type II receptor gene in NIH3T3 fibroblast cells

    SciTech Connect

    Lee, Eun Kyung; Lee, Youn Sook; Han, In-Oc; Park, Seok Hee . E-mail: parks@skku.edu

    2007-07-27

    Transcriptional repression of Transforming Growth Factor-{beta} type II receptor (T{beta}RII) gene has been proposed to be one of the major mechanisms leading to TGF-{beta} resistance. In this study, we demonstrate that expression of Caveolin-1 (Cav-1) gene in NIH3T3 fibroblast cells down-regulates the expression of T{beta}RII gene in the transcriptional level, eventually resulting in the decreased responses to TGF-{beta}. The reduced expression of T{beta}RII gene by Cav-1 appeared to be due to the changes of the sequence-specific DNA binding proteins to either Positive Regulatory Element 1 (PRE1) or PRE2 of the T{beta}RII promoter. In addition, Cav-1 expression inhibited TGF-{beta}-mediated cellular proliferation and Plasminogen Activator Inhibitor (PAI)-1 gene expression as well as TGF-{beta}-induced luciferase activity. Furthermore, the inhibition of endogeneous Cav-1 by small interfering RNA increased the expression of T{beta}RII gene. These findings strongly suggest that expression of Cav-1 leads to the decreased cellular responsiveness to TGF-{beta} through down-regulating T{beta}RII gene expression.

  18. 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

  19. Poly(acrylic acid)-regulated Synthesis of Rod-Like Calcium Carbonate Nanoparticles for Inducing the Osteogenic Differentiation of MC3T3-E1 Cells.

    PubMed

    Yang, Wei; Yao, Chenxue; Cui, Zhengyang; Luo, Dandan; Lee, In-Seop; Yao, Juming; Chen, Cen; Kong, Xiangdong

    2016-01-01

    Calcium carbonate, especially with nanostructure, has been considered as a good candidate material for bone regeneration due to its excellent biodegradability and osteoconductivity. In this study, rod-like calcium carbonate nanoparticles (Rod-CC NPs) with desired water dispersibility were achieved with the regulation of poly (acrylic acid). Characterization results revealed that the Rod-CC NPs had an average length of 240 nm, a width of 90 nm with an average aspect ratio of 2.60 and a negative ζ-potential of -22.25 ± 0.35 mV. The degradation study illustrated the nanoparticles degraded 23% at pH 7.4 and 45% at pH 5.6 in phosphate-buffered saline (PBS) solution within three months. When cultured with MC3T3-E1 cells, the Rod-CC NPs exhibited a positive effect on the proliferation of osteoblast cells. Alkaline phosphatase (ALP) activity assays together with the osteocalcin (OCN) and bone sialoprotein (BSP) expression observations demonstrated the nanoparticles could induce the differentiation of MC3T3-E1 cells. Our study developed well-dispersed rod-like calcium carbonate nanoparticles which have great potential to be used in bone regeneration. PMID:27164090

  20. Anthraquinone Glycoside Aloin Induces Osteogenic Initiation of MC3T3-E1 Cells: Involvement of MAPK Mediated Wnt and Bmp Signaling.

    PubMed

    Pengjam, Yutthana; Madhyastha, Harishkumar; Madhyastha, Radha; Yamaguchi, Yuya; Nakajima, Yuichi; Maruyama, Masugi

    2016-03-01

    Osteoporosis is a bone pathology leading to increased fracture risk and challenging the quality of life. The aim of this study was to evaluate the effect of an anthraquinone glycoside, aloin, on osteogenic induction of MC3T3-E1 cells. Aloin increased alkaline phosphatase (ALP) activity, an early differentiation marker of osteoblasts. Aloin also increased the ALP activity in adult human adipose-derived stem cells (hADSC), indicating that the action of aloin was not cell-type specific.Alizarin red S staining revealed a signifiant amount of calcium deposition in cells treated with aloin. Aloin enhanced the expression of osteoblast differentiation genes, Bmp-2, Runx2 and collagen 1a, in a dose-dependent manner. Western blot analysis revealed that noggin and inhibitors of p38 MAPK and SAPK/JNK signals attenuated aloin-promoted expressions of Bmp-2 and Runx2 proteins. siRNA mediated blocking of Wnt-5a signaling pathway also annulled the influenceof aloin, indicating Wnt-5a dependent activity. Inhibition of the different signal pathways abrogated the influenceof aloin on ALP activity, confirmingthat aloin induced MC3T3-E1 cells into osteoblasts through MAPK mediated Wnt and Bmp signaling pathway. PMID:26869456

  1. Anthraquinone Glycoside Aloin Induces Osteogenic Initiation of MC3T3-E1 Cells: Involvement of MAPK Mediated Wnt and Bmp Signaling

    PubMed Central

    Pengjam, Yutthana; Madhyastha, Harishkumar; Madhyastha, Radha; Yamaguchi, Yuya; Nakajima, Yuichi; Maruyama, Masugi

    2016-01-01

    Osteoporosis is a bone pathology leading to increased fracture risk and challenging the quality of life. The aim of this study was to evaluate the effect of an anthraquinone glycoside, aloin, on osteogenic induction of MC3T3-E1 cells. Aloin increased alkaline phosphatase (ALP) activity, an early differentiation marker of osteoblasts. Aloin also increased the ALP activity in adult human adipose-derived stem cells (hADSC), indicating that the action of aloin was not cell-type specific. Alizarin red S staining revealed a significant amount of calcium deposition in cells treated with aloin. Aloin enhanced the expression of osteoblast differentiation genes, Bmp-2, Runx2 and collagen 1a, in a dose-dependent manner. Western blot analysis revealed that noggin and inhibitors of p38 MAPK and SAPK/JNK signals attenuated aloin-promoted expressions of Bmp-2 and Runx2 proteins. siRNA mediated blocking of Wnt-5a signaling pathway also annulled the influence of aloin, indicating Wnt-5a dependent activity. Inhibition of the different signal pathways abrogated the influence of aloin on ALP activity, confirming that aloin induced MC3T3-E1 cells into osteoblasts through MAPK mediated Wnt and Bmp signaling pathway. PMID:26869456

  2. Poly(acrylic acid)-regulated Synthesis of Rod-Like Calcium Carbonate Nanoparticles for Inducing the Osteogenic Differentiation of MC3T3-E1 Cells

    PubMed Central

    Yang, Wei; Yao, Chenxue; Cui, Zhengyang; Luo, Dandan; Lee, In-Seop; Yao, Juming; Chen, Cen; Kong, Xiangdong

    2016-01-01

    Calcium carbonate, especially with nanostructure, has been considered as a good candidate material for bone regeneration due to its excellent biodegradability and osteoconductivity. In this study, rod-like calcium carbonate nanoparticles (Rod-CC NPs) with desired water dispersibility were achieved with the regulation of poly (acrylic acid). Characterization results revealed that the Rod-CC NPs had an average length of 240 nm, a width of 90 nm with an average aspect ratio of 2.60 and a negative ζ-potential of −22.25 ± 0.35 mV. The degradation study illustrated the nanoparticles degraded 23% at pH 7.4 and 45% at pH 5.6 in phosphate-buffered saline (PBS) solution within three months. When cultured with MC3T3-E1 cells, the Rod-CC NPs exhibited a positive effect on the proliferation of osteoblast cells. Alkaline phosphatase (ALP) activity assays together with the osteocalcin (OCN) and bone sialoprotein (BSP) expression observations demonstrated the nanoparticles could induce the differentiation of MC3T3-E1 cells. Our study developed well-dispersed rod-like calcium carbonate nanoparticles which have great potential to be used in bone regeneration. PMID:27164090

  3. Effect of neonatal or adult heat acclimation on plasma fT3 level, testicular thyroid receptors expression in male rats and testicular steroidogenesis in vitro in response to triiodothyronine treatment.

    PubMed

    Kurowicka, B; Chrusciel, M; Zmijewska, A; Kotwica, G

    2016-01-01

    This study aimed to evaluate the effect of heat acclimation of neonatal and adult rats on their testes response to in vitro treatment with triiodothyronine (T3). Four groups of rats were housed from birth as: 1) control (CR) at 20°C for 90 days, 2) neonatal heat-acclimated (NHA) at 34°C for 90 days, 3) adult heat-acclimated (AHA) at 20°C for 45 days followed by 45 days at 34°C and 4) de-acclimated (DA) at 34°C for 45 days followed by 45 days at 20°C. Blood plasma and both testes were harvested from 90-day old rats. Testicular slices were then submitted to in vitro treatment with T3 (100 ng/ml) for 8 h. Plasma fT3 level was lower in AHA, NHA and DA groups than in CR group. Basal thyroid hormone receptor α1 (Thra1) expression was higher in testes of NHA and DA and β1 receptor (Thrb1) in DA rats vs. other groups. In the in vitro experiment, T3: 1) decreased Thra1 expression in all groups and Thrb1 in DA group, 2) increased Star expression in CR, NHA and DA groups, and Hsd17b3 expression in NHA group, 3) decreased the expression of Cyp11a1 in NHA and DA groups, and Cyp19a1 in all the groups, 4) did not affect the activity of steroidogenic enzymes and steroid secretion (A4, T, E2) in all the groups. These results indicate, that heat acclimation of rats, depending on their age, mainly affects the testicular expression of steroidogenic enzymes in response to short-lasting treatment with T3. PMID:27487513

  4. HSP110 expression is induced by cadmium exposure but is dispensable for cell survival of mouse NIH3T3 fibroblasts.

    PubMed

    Ridley, Wakako; Nishitai, Gen; Matsuoka, Masato

    2010-05-01

    The effects of cadmium exposure on the expression of HSP110 were examined in mouse NIH3T3 fibroblasts. Following exposure to cadmium chloride, the level of HSP110 and HSP70 proteins increased after 3h and remained elevated at 24h. Similarly, their mRNA levels increased markedly in response to cadmium exposure. Treatment with 10μM mercury chloride, another toxic metal compound, also induced expression of HSP110; however, HSP110 expression was not induced in cells exposed to the same concentration of manganese chloride, zinc chloride, or lead chloride for 6 or 24h. Silencing of HSP110 expression using short-interference RNA did not affect cadmium-induced cellular damage. These results show that cadmium exposure induces the expression of high molecular weight chaperone HSP110 as well as the well-known HSP70, but indicate that HSP110 does not play a major role in cell survival following cadmium exposure. PMID:21787611

  5. A transmission electron microscopy study of constituent-particle-induced corrosion in 7075-T6 and 2024-T3 aluminum alloys

    SciTech Connect

    Wei, R.P.; Liao, C.M.; Gao, M.

    1998-04-01

    To better understand particle-induced pitting corrosion in aluminum alloys, thin foil specimens of 7075-T6 and 2024-T3 aluminum alloys, with identified constituent particles, were immersed in aerated 0.5M NaCl solution and then examined by transmission electron microscopy (TEM). The results clearly showed matrix dissolution around the iron- and manganese-containing particles (such as Al{sub 23}CuFe{sub 4}), as well as the Al{sub 2}Cu particles. While Al{sub 2}CuMg particles tended to dissolve relative to the matrix, limited local dissolution of the matrix was also observed around these particles. These results are consistent with scanning electron microscopy (SEM) observations of pitting corrosion and are discussed in terms of the electrochemical characteristics of the particles and the matrix.

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

    PubMed

    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

  7. 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

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

    NASA Astrophysics Data System (ADS)

    Cariveau, Mickael J.

    2005-07-01

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

  9. SPARC is over-expressed in adipose tissues of diet-induced obese rats and causes insulin resistance in 3T3-L1 adipocytes.

    PubMed

    Shen, Yang; Zhao, Yuyan; Yuan, Lizhi; Yi, Wei; Zhao, Rui; Yi, Qianru; Yong, Tongwu

    2014-01-01

    Secreted protein acidic and rich in cysteine (SPARC) is a secretory multifunctional matricellular glycoprotein. High circulating levels of SPARC have been reported to be associated with obesity and insulin resistance. The aim of the present study was to investigate whether SPARC induces insulin resistance and mitochondrial dysfunction in adipocytes. Our results showed that feeding high fat diet to rats for 12 weeks significantly increased SPARC expression in adipose tissues at both mRNA and protein levels. Moreover, SPARC overexpression in stably transfected 3T3-L1 cells induced insulin resistance and mitochondrial dysfunction, as evidenced by inhibition of insulin-stimulated glucose transport, lower ATP synthesis and mitochondrial membrane potential, reduced expression of glucose transporter 4 (GLUT4), and increased levels of reactive oxygen species (ROS) in mature adipocytes. Finally, overexpression of SPARC also modulated the expression levels of several inflammatory cytokines, which play important roles in insulin resistance, glucose and lipid metabolism during adipogenesis. In conclusion, our data suggest that SPARC is involved in obesity-induced adipose insulin resistance and may serve as a potential target in the treatment of obesity and obesity-related insulin resistance. PMID:23910024

  10. 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

  11. Single-cell analysis of the mitogen-induced calcium responses of normal and protein kinase C-depleted Swiss 3T3 cells.

    PubMed Central

    Corps, A N; Cheek, T R; Moreton, R B; Berridge, M J; Brown, K D

    1989-01-01

    Single-cell fluorescence image analysis has been used to characterize the mitogen-induced increases in intracellular free [Ca2+] ([Ca2+]i) in control and protein kinase C-depleted Swiss 3T3 cells. More than 80% of the control cells exhibited fast, transient responses to bombesin, vasopressin, or prostaglandin F2 alpha (PGF2 alpha). In contrast, the [Ca2+]i responses induced by platelet-derived growth factor (PDGF) were markedly more heterogeneous, slower, and often biphasic, with fewer cells (60-70%) responding. The peak [Ca2+]i values obtained in response to each mitogen showed substantial variation between cells. Brief pretreatment of the cells with 12-O-tetradecanoyl phorbol 13-acetate (TPA) reduced the [Ca2+]i responses to bombesin, but did not affect the responses to PDGF. Long-term pretreatment of the cells with TPA to down-modulate protein kinase C resulted in substantially prolonged [Ca2+]i responses to bombesin, vasopressin, and PGF2 alpha, but had no such effect on the responses to PDGF. We conclude that differences between the [Ca2+]i responses to bombesin and PDGF, previously reported using cell populations, reflect differences occurring in individual cells, and that the [Ca2+]i responses to bombesin, vasopressin, and PGF2 alpha (but not PDGF) are subject to feedback inhibition via protein kinase C. Images PMID:2519620

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

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

  13. Anti-Obesity Effects of Soy Leaf via Regulation of Adipogenic Transcription Factors and Fat Oxidation in Diet-Induced Obese Mice and 3T3-L1 Adipocytes.

    PubMed

    Li, Hua; Kang, Ji-Hyun; Han, Jong-Min; Cho, Moon-Hee; Chung, Young-Jin; Park, Ki Hun; Shin, Dong-Ha; Park, Ho-Yong; Choi, Myung-Sook; Jeong, Tae-Sook

    2015-08-01

    The anti-obesity effects of extracts from soy leaves (SLE) cultivated for 8 weeks (8W) or 16 weeks (16W) were investigated in diet-induced obese mice. The effects of kaempferol, an aglycone of the kaempferol glycosides that are the major component of 8W-SLE, and coumestrol, the major component of 16W-SLE, were also investigated in 3T3-L1 adipocytes. Eight-week-old male C57BL/6J mice were randomly divided into normal diet, high-fat diet (HFD), 8W-SLE (HFD+8W-SLE 50 mg kg(-1) day(-1)), 16W-SLE (HFD+16W-SLE 50 mg kg(-1) day(-1)), and Garcinia cambogia extracts (GE) (HFD+GE 50 mg kg(-1) day(-1)) groups. Body weight gain and fat accumulation of white adipose tissue (WAT) were highly suppressed by daily oral administration of 8W-SLE and 16W-SLE for 10 weeks. Supplementing a HFD with 8W-SLE and 16W-SLE regulated the mRNA expression of peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein alpha (c/EBPα), sterol regulatory element-binding protein-1 (SREBP-1), adipocyte protein 2, and fatty acid synthase (FAS), which are related to adipogenesis, in addition to hormone-sensitive lipase (HSL), carnitine palmitoyl transferase 1 (CPT-1), and uncoupling protein 2 (UCP2), which are related to fat oxidation in WAT. In 3T3-L1 adipocytes, kaempferol and coumestrol exhibited anti-adipogenic effects via downregulation of PPARγ, c/EBPα, SREBP-1, and FAS. Kaempferol and coumestrol increased the expression of HSL, CPT-1, and UCP2. PMID:25826408

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

    SciTech Connect

    Burch, R.M.; Axelrod, J.

    1987-09-01

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

  15. 3T3 fibroblasts induce cloned interleukin 3-dependent mouse mast cells to resemble connective tissue mast cells in granular constituency

    SciTech Connect

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

    1988-01-01

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

  16. Activation of the Ras/mitogen-activated protein kinase signaling pathway alone is not sufficient to induce glucose uptake in 3T3-L1 adipocytes.

    PubMed Central

    van den Berghe, N; Ouwens, D M; Maassen, J A; van Mackelenbergh, M G; Sips, H C; Krans, H M

    1994-01-01

    The signal transduction pathway by which insulin stimulates glucose transport is largely unknown, but a role for tyrosine and serine/threonine kinases has been proposed. Since mitogen-activated protein (MAP) kinase is activated by insulin through phosphorylation on both tyrosine and threonine residues, we investigated whether MAP kinase and its upstream regulator, p21ras, are involved in insulin-mediated glucose transport. We did this by examining the time- and dose-dependent stimulation of glucose uptake in relation to the activation of Ras-GTP formation and MAP kinase by thrombin, epidermal growth factor (EGF), and insulin in 3T3-L1 adipocytes. Ras-GTP formation was stimulated transiently by all three agonists, with a peak at 5 to 10 min. Thrombin induced a second peak at approximately 30 min. The activation of p21ras was paralleled by both the phosphorylation and the activation of MAP kinase: transient for insulin and EGF and biphasic for thrombin. However, despite the strong activation of Ras-GTP formation and MAP kinase by EGF and thrombin, glucose uptake was not stimulated by these agonists, in contrast to the eightfold stimulation of 2-deoxy-D-[14C]glucose uptake by insulin. In addition, insulin-mediated glucose transport was not potentiated by thrombin or EGF. Although these results cannot exclude the possibility that p21ras and/or MAP kinase is needed in conjunction with other signaling molecules that are activated by insulin and not by thrombin or EGF, they show that the Ras/MAP kinase signaling pathway alone is not sufficient to induce insulin-mediated glucose transport. Images PMID:7511205

  17. I lost weight, but I became weak and cannot walk--a case of nutraceutical (T3)-induced thyrotoxic periodic paralysis.

    PubMed

    Panikkath, Ragesh; Nugent, Kenneth

    2014-01-01

    Thyrotoxic periodic paralysis (TPP) is a rare reversible cause of paralysis and cramping. TPP is usually precipitated by common causes of thyrotoxicosis such as Grave disease or multinodular goiter. TPP precipitated by exogenous triiodothyronine (T3) intake is an extremely rare occurrence with only 3 cases reported to date. We now report a 24-year-old healthy manual laborer who developed quadriparesis during a period of rest after heavy exertion and carbohydrate intake. He had severe hypokalemia (potassium level 1.9 mmole/L). Correction of his hypokalemia reversed the paralysis without rebound hyperkalemia. After a detailed history review, he reported that he had been consuming nutraceuticals containing T3 for 1 month to lose weight, and laboratory studies confirmed factitious T3 toxicosis. There was no evidence of renal or gastrointestinal potassium wasting. This episode of TPP was the first manifestation of thyrotoxicosis in this patient, and avoidance of T3 intake prevented more episodes. PMID:23567793

  18. 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

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

    PubMed

    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

  20. Extracellular calcium-sensing-receptor (CaR)-mediated opening of an outward K(+) channel in murine MC3T3-E1 osteoblastic cells: evidence for expression of a functional CaR

    NASA Technical Reports Server (NTRS)

    Ye, C. P.; Yamaguchi, T.; Chattopadhyay, N.; Sanders, J. L.; Vassilev, P. M.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    2000-01-01

    The existence in osteoblasts of the G-protein-coupled extracellular calcium (Ca(o)(2+))-sensing receptor (CaR) that was originally cloned from parathyroid and kidney remains controversial. In our recent studies, we utilized multiple detection methods to demonstrate the expression of CaR transcripts and protein in several osteoblastic cell lines, including murine MC3T3-E1 cells. Although we and others have shown that high Ca(o)(2+) and other polycationic CaR agonists modulate the function of MC3T3-E1 cells, none of these actions has been unequivocally shown to be mediated by the CaR. Previous investigations using neurons and lens epithelial cells have shown that activation of the CaR stimulates Ca(2+)-activated K(+) channels. Because osteoblastic cells express a similar type of channel, we have examined the effects of specific "calcimimetic" CaR activators on the activity of a Ca(2+)-activated K(+) channel in MC3T3-E1 cells as a way of showing that the CaR is not only expressed in those cells but is functionally active. Patch-clamp analysis in the cell-attached mode showed that raising Ca(o)(2+) from 0.75 to 2.75 mmol/L elicited about a fourfold increase in the open state probability (P(o)) of an outward K(+) channel with a conductance of approximately 92 pS. The selective calcimimetic CaR activator, NPS R-467 (0.5 micromol/L), evoked a similar activation of the channel, while its less active stereoisomer, NPSS-467 (0.5 micromol/L), did not. Thus, the CaR is not only expressed in MC3T3-E1 cells, but is also functionally coupled to the activity of a Ca(2+)-activated K(+) channel. This receptor, therefore, could transduce local or systemic changes in Ca(o)(2+) into changes in the activity of this ion channel and related physiological processes in these and perhaps other osteoblastic cells.

  1. Overexpression of the beta 1 thyroid receptor induces differentiation in neuro-2a cells.

    PubMed Central

    Lebel, J M; Dussault, J H; Puymirat, J

    1994-01-01

    To determine the functions of the alpha 1 and beta 1 thyroid hormone receptors (TRs) in neural differentiation, we have established stable transfected neuronal cell lines (Neuro-2a) that overexpress either TR alpha 1 or TR beta 1. 3,5,3'-Triiodothyronine (T3) treatment of cells that overexpress TR beta 1 blocks proliferation by an arrest of cells in G0/G1 and induces morphological and functional differentiation of Neuro-2a cells as indicated by the marked increase in the number of perisomatal filopodia-like neurites and in acetylcholinesterase (AChE) activity. The effect on AChE activity was dose-dependent, and the time-course analysis reveals that this effect occurs after 24 hr of T3 treatment, with a maximal increase occurring after 48 hr of treatment. The increase of AChE activity is paralleled by an increase of AChE mRNAs. Last, we present evidence that shows that the effects of T3 on differentiation are independent of its effect on proliferation. T3 had no effect on the differentiation of Neuro-2a cells that overexpressed TR alpha 1. Our results indicate that TR beta 1 may play a key role in the effects of T3 in neuroblastoma cell differentiation. Images PMID:8146169

  2. Inhibition of MMP-13 prevents diet-induced obesity in mice and suppresses adipogenesis in 3T3-L1 preadipocytes.

    PubMed

    Shih, Chia-Li M; Ajuwon, Kolapo M

    2015-07-01

    Adipose tissue remodeling by the matrix metalloproteases (MMPs) is critical for tissue hypertrophy and obesity. MMP-13 is an important protein that is highly expressed in adipose tissue but whose potential role in adipose tissue expansion is poorly characterized. We investigated the effect of pharmacological inhibition of MMP-13 with a selective inhibitor, CP-544439, on adipose tissue mass in mice on a high fat diet, and determined the effect of the inhibitor during in vitro adipocyte differentiation of 3T3-L1 cells. CP-544439 was administered for 6 weeks to mice on a high fat diet. Body adiposity and glucose tolerance was determined. Differentiating 3T3-L1 adipocytes were also treated with the inhibitor for a maximum of 8 days and adipogenesis assessed. Treatment of mice with the inhibitor resulted in reduction in body adiposity and improvement in glucose clearance. Histological examination of epididymal adipose showed reduced adipocyte hypertrophy accompanied by increased staining for collagen in the inhibitor treated mice. Treatment of differentiating 3T3-L1 cells with the inhibitor resulted in reduced adipocyte differentiation. Knockdown of MMP-13 using small interfering RNA in differentiating 3T3-L1 cells reduced adipocyte differentiation indicated by reduced expression of PPARγ. These results suggest that MMP-13 may play a major role in adipose development and its inhibition could be a potential strategy to prevent obesity. PMID:25682268

  3. Telmisartan Induced Inhibition of Vascular Cell Proliferation beyond Angiotensin Receptor Blockade and PPARγ Activation

    PubMed Central

    Yamamoto, Koichi; Ohishi, Mitsuru; Ho, Christopher; Kurtz, Theodore W; Rakugi, Hiromi

    2010-01-01

    We investigated the ability of ARBs with PPARγ agonist activity (telmisartan and irbesartan), and ARBs devoid of PPARγ agonist activity (eprosartan and valsartan), to inhibit vascular cell proliferation studied in the absence of angiotensin II stimulation. Telmisartan and to a lesser extent irbesartan, inhibited proliferation of human aortic vascular smooth muscle cells in a dose dependent fashion whereas eprosartan and valsartan did not. To investigate the role of PPARγ in the antiproliferative effects of telmisartan, we studied genetically engineered NIH3T3 cells that express PPARγ. Pioglitazone inhibited proliferation of NIH3T3 cells expressing PPARγ, but had little effect on control NIH3T3 cells that lack PPARγ. In contrast, telmisartan inhibited proliferation equally in NIH3T3 with and without PPARγ. Valsartan failed to inhibit proliferation of either cell line. In addition, telmisartan inhibited proliferation equally in aortic smooth muscle cells derived from mice with targeted knockout of PPARγ in smooth muscle and from control mice whereas valsartan had no effect on cell proliferation. Telmisartan but not valsartan, reduced phosphorylation of AKT but not ERK otherwise induced by exposure to serum of either quiescent human smooth muscle cells, quiescent mice smooth muscle cells lacking PPARγ or quiescent CHO-K1 cells lacking AT1 receptor. In summary, the antiproliferative effect of telmisartan in the absence of exogenously supplemented angiotensin II involve more than just AT1 receptor blockade and do not require activation of PPARγ. It might be postulated that inhibition of AKT activation is a mechanism mediating the antiproliferative effects of telmisartan including in cells lacking AT1 receptors or PPARγ. PMID:19822796

  4. T3 enhances Ang2 in rat aorta in myocardial I/R: comparison with left ventricle.

    PubMed

    Sabatino, Laura; Kusmic, Claudia; Nicolini, Giuseppina; Amato, Rosario; Casini, Giovanni; Iervasi, Giorgio; Balzan, Silvana

    2016-10-01

    Angiogenesis is important for recovery after tissue damage in myocardial ischemia/reperfusion, and tri-iodothyronine (T3) has documented effects on angiogenesis. The angiopoietins 1/2 and tyrosine kinase receptor represent an essential system in angiogenesis controlling endothelial cell survival and vascular maturation. Recently, in a 3-day ischemia/reperfusion rat model, the infusion of a low dose of T3 improved the post-ischemic recovery of cardiac function.Adopting this model, our study aimed to investigate the effects of T3 on the capillary index and the expression of angiogenic genes as the angiopoietins 1/2 and tyrosine kinase receptor system, in the thoracic aorta and in the left ventricle. In the thoracic aorta, T3 infusion significantly improved the angiogenic sprouting and angiopoietin 2 expression. Instead, Sham-T3 group did not show any significant increment of capillary density and angiopoietin 2 expression. In the area at risk (AAR) of the left ventricle, T3 infusion did not increase capillary density but restored levels of angiopoietin 1, which were reduced in I/R group. Angiopoietin 2 levels were similar to Sham group and unchanged by T3 administration. In the remote zone, T3 induced a significant increment of both angiopoietin 1/2. In conclusion, T3 infusion induced a different response of angiopoietin 1/2 between the ventricle (the AAR and the remote zone) and the thoracic aorta, probably reflecting the different action of angiopoietin 1/2 in cardiomyocytes and endothelial cells. Overall, these data suggest a new aspect of T3-mediated cardioprotection through angiogenesis. PMID:27444191

  5. Effect of quinupristin/dalfopristin on 3T3 and Eahy926 cells in vitro in comparison to other antimicrobial agents with the potential to induce infusion phlebitis.

    PubMed

    Kruse, Matthias; Kilic, Bülent; Flick, Burkhard; Stahlmann, Ralf

    2007-06-01

    Infusion phlebitis is a common clinical problem that is observed with some antimicrobial agents, when being administered intravenously. In this study, cultured murine fibroblasts and immortalised human endothelial cells were exposed to three antibiotics at clinically relevant concentrations to assess their toxic potential in two established cytotoxicity assays. BALB/c 3T3 fibroblasts and Eahy926 endothelial cells were exposed to quinupristin/dalfopristin (QD), erythromycin and levofloxacin at increasing concentrations. For assessment of cytotoxicity the cells were incubated with neutral red (NR) or stained with crystal violet (CV). Measurements were done by photometry. At the concentration range tested QD and erythromycin showed a concentration-dependent cytotoxic effect in both cell cultures. In 3T3 cells the half-maximal effect concentration (EC50) was 20 mg/l for QD and 340 mg/l for erythromycin in the NR uptake test and 12 and 200 mg/l, respectively, in the CV assay. In Eahy926 cells the EC50 was 50 mg/l for QD and 880 mg/l for erythromycin in the NR uptake test and 40 and 750 mg/l, respectively, in the CV assay. No EC50 could be established in both cell types for levofloxacin. Eahy926 cells were less sensitive to cytotoxic stimuli than 3T3 fibroblasts. Cytotoxic effects in both cell cultures occurred in the following order: QD > erythromycin > levofloxacin. This ranking correlates well with the frequency of local adverse effects observed with the infusion of these antibiotics in patients. Thus, these in vitro assays may serve as an estimate for the prediction of local tolerability of antibiotics when administered parenterally. PMID:17119926

  6. Cellular regulation of poly ADP-ribosylation of proteins: II. Augmentation of poly(ADP-ribose) polymerase in SV40 3T3 cells following methotrexate-induced G1/S inhibition of cell cycle progression

    SciTech Connect

    Sooki-Toth, A.; Asghari, F.; Kirsten, E.; Kun, E. )

    1987-05-01

    SV40-3T3 cells were exposed in monolayer cultures to 5{times}10{sup {minus}7} M methotrexate (MTX), that inhibited thymidylate synthetase, arrested cell growth without cell killing in 24 h and did not induce single- (ss) or double-strand (ds) breaks in DNA. Following 24, up to 72 h, the poly(ADP-ribose) polymerase content of attached cells was induced by 5{times}10{sup {minus}7} MTX and the augmentation of the enzyme increased with the time of exposure to the drug. Inhibition of protein or RNA synthesis abolished augmentation of enzymatic activity; so too did the initiation of maximal cell growth by thymidine + hypoxanthine, by-passing the inhibitory site of MTX. Isolation of the ADP-ribosylated enzyme protein by gel electrophoresis identified poly(ADP-ribose) polymerase protein as the molecule that was induced by 5{times}10{sup {minus}7} M MTX. Under identical conditions, the poly(ADP-ribose) polymerase induction in 3T3 cells could not be demonstrated. A possible cell-cycle dependent biosynthesis of the enzyme protein is proposed in SV40 3T3 cells.

  7. Protection by the flavonoids quercetin and luteolin against peroxide- or menadione-induced oxidative stress in MC3T3-E1 osteoblast cells.

    PubMed

    Fatokun, Amos A; Tome, Mercedes; Smith, Robert A; Darlington, L Gail; Stone, Trevor W

    2015-01-01

    Potential protective effects of the flavonoids quercetin and luteolin have been examined against the oxidative stress of MC3T3-E1 osteoblast-like cells. Although hydrogen peroxide and menadione reduced cell viability, the toxicity was prevented by desferrioxamine or catalase but not superoxide dismutase, suggesting the involvement of hydrogen peroxide in both cases. Quercetin and luteolin reduced the oxidative damage, especially that caused by hydrogen peroxide. When cultures were pre-incubated with quercetin or luteolin, protection was reduced or lost. Protection was also reduced when a 24 h pre-incubation with the flavonoids was followed by exposure to menadione alone. Pretreating cultures with luteolin impaired protection by quercetin, whereas quercetin pretreatment did not affect protection by luteolin. It is concluded that quercetin and luteolin suppress oxidative damage to MC3T3-E1 cells, especially caused by peroxide. The reduction in protection by pretreatment implies a down-regulation of part of the toxic transduction pathway. PMID:25427161

  8. Amaranth lunasin-like peptide internalizes into the cell nucleus and inhibits chemical carcinogen-induced transformation of NIH-3T3 cells.

    PubMed

    Maldonado-Cervantes, Enrique; Jeong, Hyung Jin; León-Galván, Fabiola; Barrera-Pacheco, Alberto; De León-Rodríguez, Antonio; González de Mejia, Elvira; de Lumen, Ben O; Barba de la Rosa, Ana P

    2010-09-01

    Because an unbalanced diet is an important risk factor for several illnesses, interest has increased in finding novel health-promoting foods. Amaranth produces seeds that not only have substantial nutritional properties but that also contain phytochemical compounds as rutin and nicotiflorin and peptides with antihypertensive and anticarcinogenic activities. We report that a cancer-preventive peptide in amaranth has activities similar to those of soybean lunasin. The amaranth lunasin-like peptide, however, requires less time than the soybean lunasin to internalize into the nucleus of NIH-3T3 cells, and inhibits histone acetylation (H(3) and H(4) in a 70 and 77%, respectively). The amaranth lunasin-like peptide inhibited the transformation of NIH-3T3 cells to cancerous foci. The open reading frame (ORF) of amaranth lunasin corresponds to a bifunctional inhibitor/lipid-transfer protein (LTP). LTPs are a family of proteins that in plants are implicated in different functions, albeit all linked to developmental processes and biotic and abiotic stress resistance. Our results open new intriguing questions about the function of lunasin in plants and support that amaranth is a food alternative containing natural peptides with health-promoting benefits. PMID:20599579

  9. L-4F Inhibits Oxidized Low-density Lipoprotein-induced Inflammatory Adipokine Secretion via Cyclic AMP/Protein Kinase A-CCAAT/Enhancer Binding Protein β Signaling Pathway in 3T3-L1 Adipocytes

    PubMed Central

    Xie, Xiang-Zhu; Huang, Xin; Zhao, Shui-Ping; Yu, Bi-Lian; Zhong, Qiao-Qing; Cao, Jian

    2016-01-01

    Background: Adipocytes behave like a rich source of pro-inflammatory cytokines including monocyte chemoattractant protein-1 (MCP-1). Oxidized low-density lipoprotein (oxLDL) participates in the local chronic inflammatory response, and high-density lipoprotein could counterbalance the proinflammatory function of oxLDL, but the underlying mechanism is not completely understood. This study aimed to evaluate the effect of apolipoprotein A-I mimetic peptide L-4F on the secretion and expression of MCP-1 in fully differentiated 3T3-L1 adipocytes induced by oxLDL and to elucidate the possible mechanisms. Methods: Fully differentiated 3T3-L1 adipocytes were incubated in the medium containing various concentration of L-4F (0–50 μg/ml) with oxLDL (50 μg/ml) stimulated, with/without protein kinase A (PKA) inhibitor H-89 (10 μmol/L) preincubated. The concentrations of MCP-1 in the supernatant, the mRNA expression of MCP-1, the levels of CCAAT/enhancer binding protein α (C/EBPα), and CCAAT/enhancer binding protein β (C/EBPβ) were evaluated. The monocyte chemotaxis assay was performed by micropore filter method using a modified Boyden chamber. Results: OxLDL stimulation induced a significant increase of MCP-1 expression and secretion in 3T3-L1 adipocytes, which were inhibited by L-4F preincubation in a dose-dependent manner. PKA inhibitor H-89 markedly reduced the oxLDL-induced MCP-1 expression, but no further decrease was observed when H-89 was used in combination with L-4F (50 μg/ml) (P > 0.05). OxLDL stimulation showed no significant effect on C/EBPα protein level but increased C/EBPβ protein level in a time-dependent manner. H-89 and L-4F both attenuated C/EBPβ protein level in oxLDL-induced 3T3-L1 adipocytes. Conclusions: OxLDL induces C/EBPβ protein synthesis in a time-dependent manner and enhances MCP-1 secretion and expression in 3T3-L1 adipocytes. L-4F dose-dependently counterbalances the pro-inflammatory effect of oxLDL, and cyclic AMP

  10. Morphological transformation induced by multiwall carbon nanotubes on Balb/3T3 cell model as an in vitro end point of carcinogenic potential.

    PubMed

    Ponti, Jessica; Broggi, Francesca; Mariani, Valentina; De Marzi, Laura; Colognato, Renato; Marmorato, Patrick; Gioria, Sabrina; Gilliland, Douglas; Pascual Garcìa, César; Meschini, Stefania; Stringaro, Annarita; Molinari, Agnese; Rauscher, Hubert; Rossi, François

    2013-03-01

    In this work we investigated the toxicological effects of nude and chemically functionalised (-NH(2), -OH and -COOH groups) multiwall carbon nanotubes (mwCNTs) using immortalised mouse fibroblasts cell line (Balb/3T3) as in vitro model, alternative to the use of animals, to assess basal cytotoxicity, carcinogenic potential, genotoxicity and cell interaction of nanomaterials (NM). Combining in vitro tests such as cell transformation assay and micronucleus with physicochemical and topological analysis, we obtained results showing no cytotoxicity and genotoxicity. Carcinogenic potential and mwCNTs interaction with cells were instead evident. We stressed the importance that different toxicological end points have to be considered when studying NM, therefore, assays able to detect long-term effects, such as carcinogenicity, must be taken into account together with a panel of tests able to detect more immediate effects like basal cytotoxicity or genotoxicity. PMID:22279961

  11. A small difference in the molecular structure of angiotensin II receptor blockers induces AT1 receptor-dependent and -independent beneficial effects

    PubMed Central

    Fujino, Masahiro; Miura, Shin-ichiro; Kiya, Yoshihiro; Tominaga, Yukio; Matsuo, Yoshino; Karnik, Sadashiva S; Saku, Keijiro

    2013-01-01

    Angiotensin II (Ang II) type 1 (AT1) receptor blockers (ARBs) induce multiple pharmacological beneficial effects, but not all ARBs have the same effects and the molecular mechanisms underlying their actions are not certain. In this study, irbesartan and losartan were examined because of their different molecular structures (irbesartan has a cyclopentyl group whereas losartan has a chloride group). We analyzed the binding affinity and production of inositol phosphate (IP), monocyte chemoattractant protein-1 (MCP-1) and adiponectin. Compared with losartan, irbesartan showed a significantly higher binding affinity and slower dissociation rate from the AT1 receptor and a significantly higher degree of inverse agonism and insurmountability toward IP production. These effects of irbesartan were not seen with the AT1-Y113A mutant receptor. On the basis of the molecular modeling of the ARBs–AT1 receptor complex and a mutagenesis study, the phenyl group at Tyr113 in the AT1 receptor and the cyclopentyl group of irbesartan may form a hydrophobic interaction that is stronger than the losartan–AT1 receptor interaction. Interestingly, irbesartan inhibited MCP-1 production more strongly than losartan. This effect was mediated by the inhibition of nuclear factor-kappa B activation that was independent of the AT1 receptor in the human coronary endothelial cells. In addition, irbesartan, but not losartan, induced significant adiponectin production that was mediated by peroxisome proliferator-activated receptor-γ activation in 3T3-L1 adipocytes, and this effect was not mediated by the AT1 receptor. In conclusion, irbesartan induced greater beneficial effects than losartan due to small differences between their molecular structures, and these differential effects were both dependent on and independent of the AT1 receptor. PMID:20668453

  12. HOMOLOGOUS UP-REGULATION OF THE GONADOTROPIN-RELEASING HORMONE RECEPTOR IN A T3-1 CELLS IS ASSOCIATED WITH UNCHANGED RECEPTOR MESSENGER RNA (MRNA) LEVELS AND ALTERED MRNA ACTIVITY

    EPA Science Inventory

    Depending on the concentration and duration of agonist exposure, gonadotropin-releasing hormone (GnRH) receptor number either increases or decreases in response to GnRH. he molecular basis of this regulation could involve a combination of modulation of gene transcription, RNA pro...

  13. Muscarinic M1 receptor and cannabinoid CB1 receptor do not modulate paraoxon-induced seizures

    PubMed Central

    Kow, Rebecca L; Cheng, Eugene M; Jiang, Kelly; Le, Joshua H; Stella, Nephi; Nathanson, Neil M

    2015-01-01

    One of the major signs of severe organophosphate poisoning is seizures. Previous studies have shown that both muscarinic agonist- and organophosphate-induced seizures require activation of muscarinic acetylcholine receptors in the central nervous system. Seizures induced by the muscarinic agonist pilocarpine require the M1 receptor and are modulated by cannabinoid CB1 receptors. In this study, we determined whether M1 and CB1 receptors also regulated seizures induced by the organophosphate paraoxon. We found no differences in seizures induced by paraoxon in wild-type (WT) and M1 knockout (KO) mice, indicating that in contrast to pilocarpine seizures, M1 receptors are not required for paraoxon seizures. Furthermore, we found that pilocarpine administration resulted in seizure-independent activation of ERK in the hippocampus in a M1 receptor-dependent manner, while paraoxon did not induce seizure-independent activation of ERK in the mouse hippocampus. This shows that pilocarpine and paraoxon activated M1 receptors in the hippocampus to different extents. There were no differences in seizures induced by paraoxon in WT and CB1 KO mice, and neither CB1 agonist nor antagonist administration had significant effects on paraoxon seizures, indicating that, in contrast to pilocarpine seizures, paraoxon seizures are not modulated by CB1 receptors. These results demonstrate that there are fundamental molecular differences in the regulation of seizures induced by pilocarpine and paraoxon. PMID:25692018

  14. Glucose- and Triglyceride-lowering Dietary Penta-O-galloyl-α-D-Glucose Reduces Expression of PPARγ and C/EBPα, Induces p21-Mediated G1 Phase Cell Cycle Arrest, and Inhibits Adipogenesis in 3T3-L1 Preadipocytes.

    PubMed

    Liu, X; Malki, A; Cao, Y; Li, Y; Qian, Y; Wang, X; Chen, X

    2015-05-01

    Plant polyphenols, such as hydrolysable tannins, are present in the human diet and known to exhibit anti-diabetic and anti-obesity activity. We previously reported that the representative hydrolysable tannin compound α-penta-galloyl-glucose (α-PGG) is a small molecule insulin mimetic that, like insulin, binds to insulin receptor (IR) and activates the IR-Akt-GLUT4 signaling pathway to trigger glucose transport and reduce blood glucose levels in db/db and ob/ob diabetic mice. However, its effects on adipogenesis and lipid metabolism were not known. In this study, high fat diet (HFD)-induced diabetic and obese mice were treated with α-PGG to determine its effects on blood glucose and triglycerides. 3T3-L1 preadipocytes were used as a cell model for identifying the anti-adipogenic activity of α-PGG at molecular and cellular levels as a first step in elucidating the mechanism of action of the compound. In vivo, oral administration of α-PGG significantly reduced levels of blood glucose, triglyceride, and insulin in HFD-induced diabetic/obese mice (P<0.05). In vitro, α-PGG inhibited the differentiation of 3T3-L1 preadipocytes into mature adipocytes. α-PGG suppressed the expression of positive adipogenic factors PPARγ C/EBPα and mTOR and augmented the negative adipogenic factor Pref-1. Furthermore, α-PGG induced upregulation of p21 and G1 phase cell cycle arrest. In contrast, adipogenic signaling pathways mediated by insulin, the cAMP response element binding protein (CREB) and glucocorticoid receptor (GR), were not inhibited. RNAi knockdown of p21 led to a 4-fold increase in triglyceride level in 3T3-L1 preadipocytes treated with MDI and α-PGG compared to regular preadipocytes. These results indicate, for the first time, that α-PGG is blood triglyceride- and glucose-lowering in HFD-induced obese and diabetic mice. It selectively inhibited some but not all major adipogenic pathways as well as the mTOR-p21-mediated cell cycle regulatory pathway. It is very

  15. 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.

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

    SciTech Connect

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

    2013-03-29

    Highlights: ► We examined the effects of 9-cis-RA on adipogenesis in mouse preadipocyte 3T3-L1. ► 9-cis-RA inhibited lipid accumulation in adipogenetically-induced 3T3-L1 cells. ► A RXR pan-antagonist suppressed the inhibitory effects of 9-cis-RA on adipogenesis. ► This antagonist had no effects on 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.

  17. 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.

  18. Novel synergistic mechanism for sst2 somatostatin and TNFalpha receptors to induce apoptosis: crosstalk between NF-kappaB and JNK pathways.

    PubMed

    Guillermet-Guibert, J; Saint-Laurent, N; Davenne, L; Rochaix, P; Cuvillier, O; Culler, M D; Pradayrol, L; Buscail, L; Susini, C; Bousquet, C

    2007-02-01

    Somatostatin is a multifunctional hormone that modulates cell proliferation, differentiation and apoptosis. Mechanisms for somatostatin-induced apoptosis are at present mostly unsolved. Therefore, we investigated whether somatostatin receptor subtype 2 (sst2) induces apoptosis in the nontransformed murine fibroblastic NIH3T3 cells. Somatostatin receptor subtype 2 expression induced an executioner caspase-mediated apoptosis through a tyrosine phosphatase SHP-1 (Src homology domain phosphatase-1)-dependent stimulation of nuclear factor kappa B (NF-kappaB) activity and subsequent inhibition of the mitogen-activated protein kinase JNK. Tumor necrosis factor alpha (TNFalpha) stimulated both NF-kappaB and c-Jun NH2-terminal kinase (JNK) activities, which had opposite action on cell survival. Importantly, sst2 sensitized NIH3T3 cells to TNFalpha-induced apoptosis by (1) upregulating TNFalpha receptor protein expression, and sensitizing to TNFalpha-induced caspase-8 activation; (2) enhancing TNFalpha-mediated activation of NF-kappaB, resulting in JNK inhibition and subsequent executioner caspase activation and cell death. We have here unraveled a novel signaling mechanism for a G protein-coupled receptor, which directly triggers apoptosis and crosstalks with a death receptor to enhance death ligand-induced apoptosis. PMID:16645635

  19. 2-Methoxy-4-vinylphenol can induce cell cycle arrest by blocking the hyper-phosphorylation of retinoblastoma protein in benzo[a]pyrene-treated NIH3T3 cells

    SciTech Connect

    Jeong, Jin Boo; Jeong, Hyung Jin

    2010-10-01

    Research highlights: {yields} 2M4VP activated the expression of p21 and p15 protein, and down-regulated the expression of cyclin D1 and cyclin E. {yields} 2M4VP inhibited hyper-phosphorylation of Rb protein. {yields} 2M4VP induced cell cycle arrest from G1 to S. {yields} 2M4VP inhibited hyper-proliferation of the cells in BaP-treated cells. {yields} 2M4VP induces growth arrest of BaP-treated cells by blocking hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins. -- Abstract: Benzo[a]pyrene (BaP) is an environment carcinogen that can enhance cell proliferation by disturbing the signal transduction pathways in cell cycle regulation. In this study, the effects of 2M4VP on cell proliferation, cell cycle and cell cycle regulatory proteins were studied in BaP-treated NIH 3T3 cells to establish the molecular mechanisms of 2M4VP as anti-proliferative agents. 2M4VP exerted a dose-dependent inhibitory effect on cell growth correlated with a G1 arrest. Analysis of G1 cell cycle regulators expression revealed 2M4VP increased expression of CDK inhibitor, p21Waf1/Cip1 and p15 INK4b, decreased expression of cyclin D1 and cyclin E, and inhibited kinase activities of CDK4 and CDK2. However, 2M4VP did not affect the expression of CDK4 and CDK2. Also, 2M4VP inhibited the hyper-phosphorylation of Rb induced by BaP. Our results suggest that 2M4VP induce growth arrest of BaP-treated NIH 3T3 cells by blocking the hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins.

  20. Implications of epidermal growth factor (EGF) induced egf receptor aggregation.

    PubMed Central

    Wofsy, C; Goldstein, B; Lund, K; Wiley, H S

    1992-01-01

    To investigate the role of receptor aggregation in EGF binding, we construct a mathematical model describing receptor dimerization (and higher levels of aggregation) that permits an analysis of the influence of receptor aggregation on ligand binding. We answer two questions: (a) Can Scatchard plots of EGF binding data be analyzed productively in terms of two noninteracting receptor populations with different affinities if EGF induced receptor aggregation occurs? No. If two affinities characterize aggregated and monomeric EGF receptors, we show that the Scatchard plot should have curvature characteristic of positively cooperative binding, the opposite of that observed. Thus, the interpretation that the high affinity population represents aggregated receptors and the low affinity population nonaggregated receptors is wrong. If the two populations are interpreted without reference to receptor aggregation, an important determinant of Scatchard plot shape is ignored. (b) Can a model for EGF receptor aggregation and EGF binding be consistent with the "negative curvature" (i.e., curvature characteristic of negatively cooperative binding) observed in most Scatchard plots of EGF binding data? Yes. In addition, the restrictions on the model parameters required to obtain negatively curved Scatchard plots provide new information about binding and aggregation. In particular, EGF binding to aggregated receptors must be negatively cooperative, i.e., binding to a receptor in a dimer (or higher oligomer) having one receptor already bound occurs with lower affinity than the initial binding event. A third question we consider is whether the model we present can be used to detect the presence of mechanisms other than receptor aggregation that are contributing to Scatchard plot curvature. For the membrane and cell binding data we analyzed, the best least squares fits of the model to each of the four data sets deviate systematically from the data, indicating that additional factors are

  1. QRFP-43 inhibits lipolysis by preventing ligand-induced complex formation between perilipin A, caveolin-1, the catalytic subunit of protein kinase and hormone-sensitive lipase in 3T3-L1 adipocytes.

    PubMed

    Mulumba, Mukandila; Granata, Riccarda; Marleau, Sylvie; Ong, Huy

    2015-05-01

    QRFP (RFamide) peptides are neuropeptides involved in food intake and adiposity regulation in rodents. We have previously shown that QRFP-43 (43RFa) and QRFP-26 (26RFa) inhibited isoproterenol (ISO)-induced lipolysis in adipocytes. However, the antilipolytic signaling pathways activated by QRFP peptides have not been investigated. In the present study, 3T3-L1 adipocytes were used to identify the main pathways involved in QRFP-43 decreasing ISO-induced lipolysis. Our results show that QRFP-43 reduced ISO-induced phosphorylation of perilipin A (PLIN) and hormone-sensitive lipase (HSL) on Ser660 by 43 and 25%, respectively, but increased Akt phosphorylation by 44%. However, the inhibition of phosphodiesterase 3B (PDE3B), a regulator of lipolysis activated by Akt, did not reverse the antilipolytic effect of QRFP-43. PDE3B inhibition reversed the decrease of Ser660 HSL phosphorylation associated with QRFP-43 antilipolytic effect. QRFP-43 also prevented PKC activation and ISO-induced Src kinases activation leading to the inhibition of the caveolin-1 (CAV-1) translocation on lipid droplets. Indeed, QRFP-43 attenuated phorbol 12-myristate 13-acetate-induced lipolysis and ISO-induced extracellular signal-regulated and Src kinases by 28, 37 and 48%, respectively. The attenuation of ISO-induced lipolysis by QRFP-43 was associated with a decrease of phosphorylated Ser660 HSL, PKA-catalytic (PKA-c) subunit and CAV-1 translocation on lipid droplets by 37, 50 and 46%, respectively. The decrease in ISO-induced CAV-1 and PKA-c translocation was associated with a reduction of PLIN phosphorylation by 44% in QRFP-43-treated adipocytes. These results suggest that QRFP-43 attenuated ISO-induced lipolysis by preventing the formation of an active complex on lipid droplets and the activation of Src kinases and PKC. PMID:25677823

  2. The thyroid hormone receptor β induces DNA damage and premature senescence

    PubMed Central

    Zambrano, Alberto; García-Carpizo, Verónica; Gallardo, María Esther; Villamuera, Raquel; Gómez-Ferrería, Maria Ana; Pascual, Angel; Buisine, Nicolas; Sachs, Laurent M.; Garesse, Rafael

    2014-01-01

    There is increasing evidence that the thyroid hormone (TH) receptors (THRs) can play a role in aging, cancer and degenerative diseases. In this paper, we demonstrate that binding of TH T3 (triiodothyronine) to THRB induces senescence and deoxyribonucleic acid (DNA) damage in cultured cells and in tissues of young hyperthyroid mice. T3 induces a rapid activation of ATM (ataxia telangiectasia mutated)/PRKAA (adenosine monophosphate–activated protein kinase) signal transduction and recruitment of the NRF1 (nuclear respiratory factor 1) and THRB to the promoters of genes with a key role on mitochondrial respiration. Increased respiration leads to production of mitochondrial reactive oxygen species, which in turn causes oxidative stress and DNA double-strand breaks and triggers a DNA damage response that ultimately leads to premature senescence of susceptible cells. Our findings provide a mechanism for integrating metabolic effects of THs with the tumor suppressor activity of THRB, the effect of thyroidal status on longevity, and the occurrence of tissue damage in hyperthyroidism. PMID:24395638

  3. Mediator subunit MED1 is a T3-dependent and T3-independent coactivator on the thyrotropin β gene promoter

    SciTech Connect

    Matsui, Keiji; Oda, Kasumi; Mizuta, Shumpei; Ishino, Ruri; Urahama, Norinaga; Hasegawa, Natsumi; Roeder, Robert G.; Ito, Mitsuhiro

    2013-10-11

    Highlights: •MED1 is a bona fide T3-dependent coactivator on TSHB promoter. •Mice with LxxLL-mutant MED1 have attenuated TSHβ mRNA and thyroid hormone levels. •MED1 activates TSHB promoter T3-dependently in cultured cells. •T3-dependent MED1 action is enhanced when SRC1/SRC2 or HDAC2 is downregulated. •MED1 is also a T3-independent GATA2/Pit1 coactivator on TSHB promoter. -- Abstract: The MED1 subunit of the Mediator transcriptional coregulator complex is a nuclear receptor-specific coactivator. A negative feedback mechanism of thyroid-stimulating hormone (TSH, or thyrotropin) expression in the thyrotroph in the presence of triiodothyronine (T3) is employed by liganded thyroid hormone receptor β (TRβ) on the TSHβ gene promoter, where conventional histone-modifying coactivators act as corepressors. We now provide evidence that MED1 is a ligand-dependent positive cofactor on this promoter. TSHβ gene transcription was attenuated in MED1 mutant mice in which the nuclear receptor-binding ability of MED1 was specifically disrupted. MED1 stimulated GATA2- and Pit1-mediated TSHβ gene promoter activity in a ligand-independent manner in cultured cells. MED1 also stimulated transcription from the TSHβ gene promoter in a T3-dependent manner. The transcription was further enhanced when the T3-dependent corepressors SRC1, SRC2, and HDAC2 were downregulated. Hence, MED1 is a T3-dependent and -independent coactivator on the TSHβ gene promoter.

  4. Non-NMDA receptor antagonist-induced drinking in rat

    NASA Technical Reports Server (NTRS)

    Xu, Z.; Johnson, A. K.

    1998-01-01

    Glutamate has been implicated in the central control of mechanisms that maintain body fluid homeostasis. The present studies demonstrate that intracerebroventricular (i.c.v.) injections of the non-N-methyl-d-aspartate (NMDA) receptor antagonists 6, 7-dinitroquinoxaline-2,3-dione (DNQX) and 6-cyano-7-nitroquinoxaline-2,3 dione (CNQX) induce drinking in rats. The dipsogenic effect of i.c.v. DNQX was antagonized by the non-NMDA receptor agonist alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). The water intake induced by DNQX was also blocked by pretreatment with a NMDA receptor antagonist, MK-801, but not by angiotensin type 1 (AT1) or acetylcholine muscarinic receptor antagonists (losartan and atropine). The results indicate that non-NMDA receptors may exert a tonic inhibitory effect within brain circuits that control dipsogenic activity and that functional integrity of NMDA receptors may be required for the non-NMDA receptor antagonists to induce water intake. Copyright 1998 Published by Elsevier Science B.V.

  5. Liganded Thyroid Hormone Receptor Inhibits Phorbol 12-O-Tetradecanoate-13-Acetate-Induced Enhancer Activity via Firefly Luciferase cDNA

    PubMed Central

    Misawa, Hiroko; Sasaki, Shigekazu; Matsushita, Akio; Ohba, Kenji; Iwaki, Hiroyuki; Matsunaga, Hideyuki; Suzuki, Shingo; Ishizuka, Keiko; Oki, Yutaka; Nakamura, Hirotoshi

    2012-01-01

    Thyroid hormone receptor (TR) belongs to the nuclear hormone receptor (NHR) superfamily and regulates the transcription of its target genes in a thyroid hormone (T3)-dependent manner. While the detail of transcriptional activation by T3 (positive regulation) has been clarified, the mechanism of T3-dependent repression (negative regulation) remains to be determined. In addition to naturally occurring negative regulations typically found for the thyrotropin β gene, T3-bound TR (T3/TR) is known to cause artificial negative regulation in reporter assays with cultured cells. For example, T3/TR inhibits the transcriptional activity of the reporter plasmids harboring AP-1 site derived from pUC/pBR322-related plasmid (pUC/AP-1). Artificial negative regulation has also been suggested in the reporter assay with firefly luciferase (FFL) gene. However, identification of the DNA sequence of the FFL gene using deletion analysis was not performed because negative regulation was evaluated by measuring the enzymatic activity of FFL protein. Thus, there remains the possibility that the inhibition by T3 is mediated via a DNA sequence other than FFL cDNA, for instance, pUC/AP-1 site in plasmid backbone. To investigate the function of FFL cDNA as a transcriptional regulatory sequence, we generated pBL-FFL-CAT5 by ligating FFL cDNA in the 5' upstream region to heterologous thymidine kinase promoter in pBL-CAT5, a chloramphenicol acetyl transferase (CAT)-based reporter gene, which lacks pUC/AP-1 site. In kidney-derived CV1 and choriocarcinoma-derived JEG3 cells, pBL-FFL-CAT5, but not pBL-CAT5, was strongly activated by a protein kinase C activator, phorbol 12-O-tetradecanoate-13-acetate (TPA). TPA-induced activity of pBL-FFL-CAT5 was negatively regulated by T3/TR. Mutation of nt. 626/640 in FFL cDNA attenuated the TPA-induced activation and concomitantly abolished the T3-dependent repression. Our data demonstrate that FFL cDNA sequence mediates the TPA-induced transcriptional activity

  6. Antiobesity Effects of an Edible Halophyte Nitraria retusa Forssk in 3T3-L1 Preadipocyte Differentiation and in C57B6J/L Mice Fed a High Fat Diet-Induced Obesity

    PubMed Central

    Zar Kalai, Feten; Han, Junkyu; Ksouri, Riadh; El Omri, Abdelfatteh; Abdelly, Chedly; Isoda, Hiroko

    2013-01-01

    Nitraria retusa is an edible halophyte, used in Tunisia for several traditional medicine purposes. The present study investigated the antiobesity effects of Nitraria retusa ethanol extract (NRE) in 3T3-L1 cells using different doses and in high-fat diet-induced obesity in mice. Male C57B6J/L mice were separately fed a normal diet (ND) or a high-fat diet (HFD) and daily administrated with NRE (50, 100 mg/kg) or one for 2 days with Naringenin (10 mg/kg). NRE administration significantly decreased body weight gain, fat pad weight, serum glucose, and lipid levels in HFD-induced obese mice. To elucidate the mechanism of action of NRE, the expression of genes involved in lipid and carbohydrate metabolism were measured in liver. Results showed that mice treated with NRE demonstrated a significant decrease in cumulative body weight and fat pad weight, a significant lowering in glucose and triglycerides serum levels, and an increase in the HDL-cholesterol serum level. Moreover mRNA expression results showed an enhancement of the expression of genes related to liver metabolism. Our findings suggest that NRE treatment had a protective or controlling effect against a high fat diet-induced obesity in C57B6J/L mice through the regulation of expression of genes involved in lipolysis and lipogenesis and thus the enhancement of the lipid metabolism in liver. PMID:24367387

  7. Tumor necrosis factor-induced reversal of adipocytic phenotype of 3T3-L1 cells is preceded by a loss of nuclear CCAAT/enhancer binding protein (C/EBP).

    PubMed Central

    Ron, D; Brasier, A R; McGehee, R E; Habener, J F

    1992-01-01

    Tumor necrosis factor (TNF)-treated 3T3-L1 adipocytes were used as a model for studying the effects of systemic inflammation on adipose tissue. Lipopolysaccharide-treated monocyte-conditioned medium or recombinant human TNF alpha induced morphological dedifferentiation of the adipocytes and led to loss of adipocyte specific gene expression. Gel shift, Southwestern and Western immunoblot analysis demonstrated that dedifferentiation was preceded by a decrease in the DNA binding activity and protein level of the transcription factor CCAAT/enhancer binding protein (C/EBP). Liver activating protein, a related protein that binds identical DNA sequences, increased during cytokine treatment. Both proteins activate specific enhancer elements located in the promoter region of many genes whose transcription is altered during systemic inflammation. Pulse-chase labeling followed by immunoprecipitation demonstrated that C/EBP is a rapidly turning over protein in adipocytes and that cytokine treatment led to a specific, time dependent decrease in its rate of synthesis. Because C/EBP binding sites have been shown to play an important role in regulating the expression of genes involved in adipocyte metabolism, we propose that the TNF-induced changes in the complement of transcription factors binding those sites may be important in the pathogenesis of inflammation-induced atrophy of adipose tissue. Images PMID:1729273

  8. TRPV1 receptors mediate particulate matter-induced apoptosis.

    PubMed

    Agopyan, N; Head, J; Yu, S; Simon, S A

    2004-03-01

    Exposure to airborne particulate matter (PM) is a world-wide health problem mainly because it produces adverse cardiovascular and respiratory effects that frequently result in morbidity. Despite many years of epidemiological and basic research, the mechanisms underlying PM toxicity remain largely unknown. To understand some of these mechanisms, we measured PM-induced apoptosis and necrosis in normal human airway epithelial cells and sensory neurons from both wild-type mice and mice lacking TRPV1 receptors using Alexa Fluor 488-conjugated annexin V and propidium iodide labeling, respectively. Exposure of environmental PMs containing residual oil fly ash and ash from Mount St. Helens was found to induce apoptosis, but not necrosis, as a consequence of sustained calcium influx through TRPV1 receptors. Apoptosis was completely prevented by inhibiting TRPV1 receptors with capsazepine or by removing extracellular calcium or in sensory neurons from TRPV1(-/-) mice. Binding of either one of the PMs to the cell membrane induced a capsazepine-sensitive increase in cAMP. PM-induced apoptosis was augmented upon the inhibition of PKA. PKA inhibition on its own also induced apoptosis, thereby suggesting that this pathway may be endogenously protective against apoptosis. In summary, it was found that inhibiting TRPV1 receptors prevents PM-induced apoptosis, thereby providing a potential mechanism to reduce their toxicity. PMID:14633515

  9. Peripheral endothelin A receptor antagonism attenuates carcinoma-induced pain.

    PubMed

    Schmidt, Brian L; Pickering, Victoria; Liu, Stanley; Quang, Phuong; Dolan, John; Connelly, S Thaddeus; Jordan, Richard C K

    2007-05-01

    In this study we investigated the role of endothelin-1 (ET-1) and its peripheral receptor (ET-A) in carcinoma-induced pain in a mouse cancer pain model. Tumors were induced in the hind paw of female mice by local injection of cells derived from a human oral squamous cell carcinoma (SCC). Significant pain, as indicated by reduction in withdrawal thresholds in response to mechanical stimulation, began at four days after SCC inoculation and lasted to 28 days, the last day of measurement. Intra-tumor expression of both ET-1 mRNA and ET-1 protein were significantly upregulated compared to normal tissue, and local administration of the ET-A receptor selective antagonist, BQ-123 (100 microM) significantly elevated withdrawal thresholds, indicating the induction of an antinociceptive effect. These findings support the suggestion that ET-1 and ET-A receptors contribute to the severity of carcinoma-induced soft tissue cancer pain. PMID:16807013

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

    PubMed

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

    2009-01-01

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

  11. Wnt/β-catenin signaling plays an important role in the protective effects of FDP-Sr against oxidative stress induced apoptosis in MC3T3-E1 cell.

    PubMed

    Qi, Huan-Huan; Bao, Jun; Zhang, Qi; Ma, Bo; Gu, Gui-Ying; Zhang, Peng-Ling; Ou-Yang, Gang; Wu, Zi-Mei; Ying, Han-Jie; Ou-Yang, Ping-Kai

    2016-10-01

    Strontium fructose 1,6-diphosphate (FDP-Sr) is a new strontium-containing compound. The primary aim of this study was to clarify whether the structure component of FDP-Sr, FDP could benefit the protective effect of Sr (II) against oxidative stress induced apoptosis, and meanwhile to further explore the important role of Wnt/β-catenin signaling in the anti-apoptosis effect of FDP-Sr in response to oxidative stress induced by H2O2 in an osteoblastic MC3T3-E1 cell line. Results showed that FDP-Sr could improve the osteoblastic differentiation under oxidative stress with induced cell proliferation and improved mineralization. The inhibition effect of FDP-Sr on cell apoptosis induced by H2O2 was proved by reduced reactive oxygen species production and activated caspase3. Under oxidative stress, mRNA and protein levels of phospho-β-catenin reduced, while β-catenin increased in the FDP-Sr treatment cell, leaded to the up-regulations of Runx2 and OPG at both mRNA and protein levels, finally improved the differentiation of osteoblasts. By the engagement of Wnt/β-catenin pathway's inhibitor (XAV-939), the protective effects of FDP-Sr on osteoblastic differentiation against oxidative stress were repressed along with inhibited wnt/β-catenin signaling and reduced mRNA and protein levels of Runx2 and OPG. In conclusion, FDP-Sr was demonstrated to protect osteoblast differentiation from oxidative damage induced by H2O2 through up-regulation of Wnt/β-catenin signaling, and FDP in FDP-Sr was able to directly improve the oxidative stress injury through its ROS scavenging ability. PMID:27575480

  12. Type III secretion needle proteins induce cell signaling and cytokine secretion via Toll-like receptors.

    PubMed

    Jessen, Danielle L; Osei-Owusu, Patrick; Toosky, Melody; Roughead, William; Bradley, David S; Nilles, Matthew L

    2014-06-01

    Pathogens are recognized by hosts by use of various receptors, including the Toll-like receptor (TLR) and Nod-like receptor (NLR) families. Ligands for these varied receptors, including bacterial products, are identified by the immune system, resulting in development of innate immune responses. Only a couple of components from type III secretion (T3S) systems are known to be recognized by TLR or NLR family members. Known T3S components that are detected by pattern recognition receptors (PRRs) are (i) flagellin, detected by TLR5 and NLRC4 (Ipaf); and (ii) T3S rod proteins (PrgJ and homologs) and needle proteins (PrgI and homologs), detected by NAIP and the NLRC4 inflammasome. In this report, we characterize the induction of proinflammatory responses through TLRs by the Yersinia pestis T3S needle protein, YscF, the Salmonella enterica needle proteins PrgI and SsaG, and the Shigella needle protein, MxiH. More specifically, we determine that the proinflammatory responses occur through TLR2 and -4. These data support the hypothesis that T3S needles have an unrecognized role in bacterial pathogenesis by modulating immune responses. PMID:24643544

  13. Prostaglandin E2-induced up-regulation of c-fos messenger ribonucleic acid is primarily mediated by 3',5'-cyclic adenosine monophosphate in MC3T3-E1 osteoblasts

    NASA Technical Reports Server (NTRS)

    Fitzgerald, J.; Dietz, T. J.; Hughes-Fulford, M.

    2000-01-01

    The mechanism by which the proto-oncogene, c-fos, is up-regulated in response to PGE2 in the mouse osteoblastic (MC3T3-E1) cell line was investigated using RT-PCR. c-fos messenger RNA up-regulation by dmPGE2 is rapid, starting 10 min post stimulation, and transient. The specific protein kinase A (PKA) inhibitor, H89, inhibited c-fos induction. Moreover, down-regulation of protein kinase C (PKC) activity by chronic TPA treatment had no effect on the induction of c-fos by dmPGE2. We conclude that up-regulation of c-fos by dmPGE2 is primarily dependent on PKA in MC3T3-E1 osteoblasts. In S49 lymphoma wild-type but not S49 cyc- cells, which are deficient in cAMP signaling, dmPGE2 up-regulates c-fos and increases cell growth compared with unstimulated cells. Thus in S49 lymphoma cells, c-fos induction by PGE2 is also dependent on cAMP signaling. The minimal c-fos promoter region required for dmPGE2-induced expression was identified by transfecting c-fos promoter deletion constructs coupled to the chloramphenicol acetyltransferase (CAT) reporter gene into Vero cells. Transfection of a plasmid containing 99 bp c-fos proximal promoter was sufficient to direct c-fos/CAT expression following stimulation with dmPGE2. Because induction of c-fos is mediated by cAMP, these data are consistent with activation of c-fos via the CRE/ATF cis element.

  14. Modulation of Cell-Substrate Adhesion by Arachidonic Acid: Lipoxygenase Regulates Cell Spreading and ERK1/2-inducible Cyclooxygenase Regulates Cell Migration in NIH-3T3 Fibroblasts

    PubMed Central

    Stockton, Rebecca A.; Jacobson, Bruce S.

    2001-01-01

    Adhesion of cells to an extracellular matrix is characterized by several discrete morphological and functional stages beginning with cell-substrate attachment, followed by cell spreading, migration, and immobilization. We find that although arachidonic acid release is rate-limiting in the overall process of adhesion, its oxidation by lipoxygenase and cyclooxygenases regulates, respectively, the cell spreading and cell migration stages. During the adhesion of NIH-3T3 cells to fibronectin, two functionally and kinetically distinct phases of arachidonic acid release take place. An initial transient arachidonate release occurs during cell attachment to fibronectin, and is sufficient to signal the cell spreading stage after its oxidation by 5-lipoxygenase to leukotrienes. A later sustained arachidonate release occurs during and after spreading, and signals the subsequent migration stage through its oxidation to prostaglandins by newly synthesized cyclooxygenase-2. In signaling migration, constitutively expressed cyclooxygenase-1 appears to contribute ∼25% of prostaglandins synthesized compared with the inducible cyclooxygenase-2. Both the second sustained arachidonate release, and cyclooxygenase-2 protein induction and synthesis, appear to be regulated by the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK)1/2. The initial cell attachment-induced transient arachidonic acid release that signals spreading through lipoxygenase oxidation is not sensitive to ERK1/2 inhibition by PD98059, whereas PD98059 produces both a reduction in the larger second arachidonate release and a blockade of induced cyclooxygenase-2 protein expression with concomitant reduction of prostaglandin synthesis. The second arachidonate release, and cyclooxygenase-2 expression and activity, both appear to be required for cell migration but not for the preceding stages of attachment and spreading. These data suggest a bifurcation in the arachidonic acid adhesion

  15. Amyloid-β-Induced Dysregulation of AMPA Receptor Trafficking

    PubMed Central

    Guntupalli, Sumasri; Widagdo, Jocelyn; Anggono, Victor

    2016-01-01

    Evidence from neuropathological, genetic, animal model, and biochemical studies has indicated that the accumulation of amyloid-beta (Aβ) is associated with, and probably induces, profound neuronal changes in brain regions critical for memory and cognition in the development of Alzheimer's disease (AD). There is considerable evidence that synapses are particularly vulnerable to AD, establishing synaptic dysfunction as one of the earliest events in pathogenesis, prior to neuronal loss. It is clear that excessive Aβ levels can disrupt excitatory synaptic transmission and plasticity, mainly due to dysregulation of the AMPA and NMDA glutamate receptors in the brain. Importantly, AMPA receptors are the principal glutamate receptors that mediate fast excitatory neurotransmission. This is essential for synaptic plasticity, a cellular correlate of learning and memory, which are the cognitive functions that are most disrupted in AD. Here we review recent advances in the field and provide insights into the molecular mechanisms that underlie Aβ-induced dysfunction of AMPA receptor trafficking. This review focuses primarily on NMDA receptor- and metabotropic glutamate receptor-mediated signaling. In particular, we highlight several mechanisms that underlie synaptic long-term depression as common signaling pathways that are hijacked by the neurotoxic effects of Aβ. PMID:27073700

  16. Amyloid-β-Induced Dysregulation of AMPA Receptor Trafficking.

    PubMed

    Guntupalli, Sumasri; Widagdo, Jocelyn; Anggono, Victor

    2016-01-01

    Evidence from neuropathological, genetic, animal model, and biochemical studies has indicated that the accumulation of amyloid-beta (Aβ) is associated with, and probably induces, profound neuronal changes in brain regions critical for memory and cognition in the development of Alzheimer's disease (AD). There is considerable evidence that synapses are particularly vulnerable to AD, establishing synaptic dysfunction as one of the earliest events in pathogenesis, prior to neuronal loss. It is clear that excessive Aβ levels can disrupt excitatory synaptic transmission and plasticity, mainly due to dysregulation of the AMPA and NMDA glutamate receptors in the brain. Importantly, AMPA receptors are the principal glutamate receptors that mediate fast excitatory neurotransmission. This is essential for synaptic plasticity, a cellular correlate of learning and memory, which are the cognitive functions that are most disrupted in AD. Here we review recent advances in the field and provide insights into the molecular mechanisms that underlie Aβ-induced dysfunction of AMPA receptor trafficking. This review focuses primarily on NMDA receptor- and metabotropic glutamate receptor-mediated signaling. In particular, we highlight several mechanisms that underlie synaptic long-term depression as common signaling pathways that are hijacked by the neurotoxic effects of Aβ. PMID:27073700

  17. Primary Macrophage Chemotaxis Induced by Cannabinoid Receptor 2 Agonists Occurs Independently of the CB2 Receptor

    PubMed Central

    Taylor, Lewis; Christou, Ivy; Kapellos, Theodore S.; Buchan, Alice; Brodermann, Maximillian H.; Gianella-Borradori, Matteo; Russell, Angela; Iqbal, Asif J.; Greaves, David R.

    2015-01-01

    Activation of CB2 has been demonstrated to induce directed immune cell migration. However, the ability of CB2 to act as a chemoattractant receptor in macrophages remains largely unexplored. Using a real-time chemotaxis assay and a panel of chemically diverse and widely used CB2 agonists, we set out to examine whether CB2 modulates primary murine macrophage chemotaxis. We report that of 12 agonists tested, only JWH133, HU308, L-759,656 and L-759,633 acted as macrophage chemoattractants. Surprisingly, neither pharmacological inhibition nor genetic ablation of CB2 had any effect on CB2 agonist-induced macrophage chemotaxis. As chemotaxis was pertussis toxin sensitive in both WT and CB2-/- macrophages, we concluded that a non-CB1/CB2, Gi/o-coupled GPCR must be responsible for CB2 agonist-induced macrophage migration. The obvious candidate receptors GPR18 and GPR55 could not mediate JWH133 or HU308-induced cytoskeletal rearrangement or JWH133-induced β-arrestin recruitment in cells transfected with either receptor, demonstrating that neither are the unidentified GPCR. Taken together our results conclusively demonstrate that CB2 is not a chemoattractant receptor for murine macrophages. Furthermore we show for the first time that JWH133, HU308, L-759,656 and L-759,633 have off-target effects of functional consequence in primary cells and we believe that our findings have wide ranging implications for the entire cannabinoid field. PMID:26033291

  18. Regulation of particulate matter-induced mucin secretion by transient receptor potential vanilloid 1 receptors.

    PubMed

    Yu, Hongmei; Li, Qi; Kolosov, Victor P; Perelman, Juliy M; Zhou, Xiangdong

    2012-12-01

    Exposure to airborne particulate matter (PM) is a worldwide health problem. Previous studies have reported that PMs induced depolarizing currents and increased intracellular Ca(2+) in human bronchial epithelial cells. Ca(2+) plays important role in the regulation of mucus exocytosis, and mucin hypersecretion is a key pathological feature of inflammatory respiratory diseases. To explore more mechanisms underlying PM toxicity, we measured PM-induced mucin secretion in human bronchial epithelial (16HBE) cells. MUC5AC secretion and cyclic adenosine monophosphate (cAMP) level were detected by ELISA. Transient receptor potential vanilloid (TRPV)1 inward currents were examined by electrophysiology. Ca(2+) concentration was assessed by laser scanning confocal microscope. Exposure of PMs to 16HBE cells was found to induce mucin secretion, as a consequence of sustained Ca(2+) influx and cAMP increase through TRPV1 receptors. Mucin secretion was completely inhibited by TRPV1 receptor antagonist capsazepine. Removal of Ca(2+) by Ca(2+) chelator BAPTA or inhibition of protein kinase A (PKA) by the PKA inhibitors H-89 each partially reduced PC(2)s-induced mucin secretion. The combination of BAPTA and H-89 completely prevented mucin secretion mediated by PMs. These results suggest that PM induces mucin secretion through Ca(2+) influx and cAMP/PKA pathway by TRPV1 receptors in human bronchial epithelial cells, thereby providing a potential mechanism to reduce PM toxicity. PMID:22829138

  19. Fluid shear stress suppresses TNF-α-induced apoptosis in MC3T3-E1 cells: Involvement of ERK5-AKT-FoxO3a-Bim/FasL signaling pathways.

    PubMed

    Bin, Geng; Bo, Zhang; Jing, Wang; Jin, Jiang; Xiaoyi, Tan; Cong, Chen; Liping, An; Jinglin, Ma; Cuifang, Wang; Yonggang, Chen; Yayi, Xia

    2016-05-01

    TNF-α is known to induce osteoblasts apoptosis, whereas mechanical stimulation has been shown to enhance osteoblast survival. In the present study, we found that mechanical stimulation in the form of fluid shear stress (FSS) suppresses TNF-α induced apoptosis in MC3T3-E1 cells. Extracellular signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family that has been implicated in cell survival. We also demonstrated that FSS imposed by flow chamber in vitro leads to a markedly activation of ERK5, which was shown to be protective against TNF-α-induced apoptosis, whereas the transfection of siRNA against ERK5 (ERK5-siRNA) reversed the FSS-medicated anti-apoptotic effects. An initial FSS-mediated activation of ERK5 that phosphorylates AKT to increase its activity, and a following forkhead box O 3a (FoxO3a) was phosphorylated by activated AKT. Phosphorylated FoxO3a is sequestered in the cytoplasm, and prevents it from translocating to nucleus where it can increase the expression of FasL and Bim. The inhibition of AKT-FoxO3a signalings by a PI3K (PI3-kinase)/AKT inhibitor (LY294002) or the transfection of ERK5-siRNA led to the nuclear translocation of non-phosphorylated FoxO3a, and increased the protein expression of FasL and Bim. In addition, the activation of caspase-3 by TNF-α was significantly inhibited by aforementioned FSS-medicated mechanisms. In brief, the activation of ERK5-AKT-FoxO3a signaling pathways by FSS resulted in a decreased expression of FasL and Bim and an inhibition of caspase-3 activation, which exerts a protective effect that prevents osteoblasts from apoptosis. PMID:27060196

  20. 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.

  1. 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. PMID:21031614

  2. Identification of cytoprotective constituents of the flower buds of Tussilago farfara against glucose oxidase-induced oxidative stress in mouse fibroblast NIH3T3 cells and human keratinocyte HaCaT cells.

    PubMed

    Kang, Unwoo; Park, Jiyoung; Han, Ah-Reum; Woo, Mi Hee; Lee, Je-Hyun; Lee, Sang Kook; Chang, Tong-Shin; Woo, Hyun Ae; Seo, Eun Kyoung

    2016-04-01

    A new cytoprotective compound, 1-[(4S)-3,4-dihydro-4-hydroxy-2,2-dimethyl-2H-1-benzopyran-6-yl]-ethanone (1) was isolated from the flower buds of Tussilago farfara L. (Compositae), together with eight known compounds, 3,4-dicaffeoyl isoquinic acid (2), trans-cinnamic acid (3), 4-hydroxyacetophenone (4), 4,5-dicaffeoylquinic acid methyl ester (5), 3,5-dicaffeoylquinic acid methyl ester (6), 4-hydroxybenzoic acid (7), isoquercetrin (8), and ligucyperonol (9). Compounds 2-4 were found in this plant for the first time. The isolates 1-9, were tested for their cytoprotective activities against glucose oxidase-induced oxidative stress in mouse fibroblast NIH3T3 cells and human keratinocyte HaCaT cells. Among them, 1 and 3 showed significant cytoprotective activities as determined by MTT assay and lactate dehydrogenase leakage, indicating their possibility as the potent cytoprotective agents. The structure of 1 was determined by spectroscopic data analysis including 1D- and 2D-NMR experiments, and its absolute configuration was elucidated by a circular dichroism. PMID:26983826

  3. TRPA1 receptors mediate environmental irritant-induced meningeal vasodilatation

    PubMed Central

    Kunkler, Phillip Edward; Ballard, Carrie Jo; Oxford, Gerry Stephen; Hurley, Joyce Harts

    2010-01-01

    The TRPA1 receptor is a member of the transient receptor potential (TRP) family of ion channels expressed in nociceptive neurons. TRPA1 receptors are targeted by pungent compounds from mustard and garlic and environmental irritants such as formaldehyde and acrolein. Ingestion or inhalation of these chemical agents causes irritation and burning in the nasal and oral mucosa and respiratory lining. Headaches have been widely reported to be induced by inhalation of environmental irritants, but it is unclear how these agents produce headache. Stimulation of trigeminal neurons releases CGRP and substance P and induces neurogenic inflammation associated with the pain of migraine. Here we test the hypothesis that activation of TRPA1 receptors are the mechanistic link between environmental irritants and peptide mediated neurogenic inflammation. Known TRPA1 agonists and environmental irritants stimulate CGRP release from dissociated rat trigeminal ganglia neurons and this release is blocked by a selective TRPA1 antagonist, HC-030031. Further, TRPA1 agonists and environmental irritants increase meningeal blood flow following intranasal administration. Prior dural application of the CGRP antagonist, CGRP8–37, or intranasal or dural administration of HC-030031, blocks the increases in blood flow elicited by environmental irritants. Together these results demonstrate that TRPA1 receptor activation by environmental irritants stimulates CGRP release and increases cerebral blood flow. We suggest that these events contribute to headache associated with environmental irritants. PMID:21075522

  4. Stress Induces Pain Transition by Potentiation of AMPA Receptor Phosphorylation

    PubMed Central

    Li, Changsheng; Yang, Ya; Liu, Sufang; Fang, Huaqiang; Zhang, Yong; Furmanski, Orion; Skinner, John; Xing, Ying; Johns, Roger A.

    2014-01-01

    Chronic postsurgical pain is a serious issue in clinical practice. After surgery, patients experience ongoing pain or become sensitive to incident, normally nonpainful stimulation. The intensity and duration of postsurgical pain vary. However, it is unclear how the transition from acute to chronic pain occurs. Here we showed that social defeat stress enhanced plantar incision-induced AMPA receptor GluA1 phosphorylation at the Ser831 site in the spinal cord and greatly prolonged plantar incision-induced pain. Interestingly, targeted mutation of the GluA1 phosphorylation site Ser831 significantly inhibited stress-induced prolongation of incisional pain. In addition, stress hormones enhanced GluA1 phosphorylation and AMPA receptor-mediated electrical activity in the spinal cord. Subthreshold stimulation induced spinal long-term potentiation in GluA1 phosphomimetic mutant mice, but not in wild-type mice. Therefore, spinal AMPA receptor phosphorylation contributes to the mechanisms underlying stress-induced pain transition. PMID:25297100

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

    PubMed

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

    2015-10-01

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

  6. A primer on cytokines: sources, receptors, effects, and inducers.

    PubMed Central

    Curfs, J H; Meis, J F; Hoogkamp-Korstanje, J A

    1997-01-01

    Protection against pathogens is a prerequisite for survival of most organisms. To cope with this continuous challenge, complex defense mechanisms have evolved. The construction, adaptation, and maintenance of these mechanisms are under control of an extensive network of regulatory proteins called cytokines. A great number of cytokines have been described over the last 2 decades. This review consists of an overview of cytokines that are involved in immune responses and describes some historical and general aspects as well as prospective clinical applications. Major biological effects together with information on cytokine receptors, producers, inducers, and biochemical and molecular characteristics are listed in tables. In addition, some basic information is given on cytokine receptor signal transduction. Finally, the recent discoveries of cytokine receptors functioning as coreceptors in the pathogenesis of human immunodeficiency virus are summarized. PMID:9336671

  7. Crosslinking-Induced Endocytosis of Acetylcholine Receptors by Quantum Dots

    PubMed Central

    Geng, Lin; Peng, H. Benjamin

    2014-01-01

    In a majority of patients with myasthenia gravis (MG), anti-acetylcholine receptor (AChR) antibodies target postsynaptic AChR clusters and thus compromise the membrane integrity of neuromuscular junctions (NMJs) and lead to muscle weakness. Antibody-induced endocytosis of AChRs in the postsynaptic membrane represents the initial step in the pathogenesis of MG; however, the molecular mechanisms underlying AChR endocytosis remain largely unknown. Here, we developed an approach to mimic the pathogenic antibodies for inducing the crosslinking and internalization of AChRs from the postsynaptic membrane. Using biotin-α-bungarotoxin and quantum dot (QD)-streptavidin, cell-surface and internalized AChRs could be readily distinguished by comparing the size, fluorescence intensity, trajectory, and subcellular localization of the QD signals. QD-induced AChR endocytosis was mediated by clathrin-dependent and caveolin-independent mechanisms, and the trafficking of internalized AChRs in the early endosomes required the integrity of microtubule structures. Furthermore, activation of the agrin/MuSK (muscle-specific kinase) signaling pathway strongly suppressed QD-induced internalization of AChRs. Lastly, QD-induced AChR crosslinking potentiated the dispersal of aneural AChR clusters upon synaptic induction. Taken together, our results identify a novel approach to study the mechanisms of AChR trafficking upon receptor crosslinking and endocytosis, and demonstrate that agrin-MuSK signaling pathways protect against crosslinking-induced endocytosis of AChRs. PMID:24587270

  8. 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

  9. The orphan nuclear receptor estrogen receptor-related receptor gamma negatively regulates BMP2-induced osteoblast differentiation and bone formation.

    PubMed

    Jeong, Byung-Chul; Lee, Yong-Soo; Park, Yun-Yong; Bae, In-Ho; Kim, Don-Kyu; Koo, Seung-Hoi; Choi, Hong-Ran; Kim, Sun-Hun; Franceschi, Renny T; Koh, Jeong-Tae; Choi, Hueng-Sik

    2009-05-22

    Estrogen receptor-related receptor gamma (ERRgamma/ERR3/NR3B3) is a member of the orphan nuclear receptor with important functions in development and homeostasis. Recently it has been reported that ERRalpha is involved in osteoblast differentiation and bone formation. In the present study we examined the role of ERRgamma in osteoblast differentiation. Here, we showed that ERRgamma is expressed in osteoblast progenitors and primary osteoblasts, and its expression is increased temporarily by BMP2. Overexpression of ERRgamma reduced BMP2-induced alkaline phosphatase activity and osteocalcin production as well as calcified nodule formation, whereas inhibition of ERRgamma expression significantly enhanced BMP2-induced osteogenic differentiation and mineralization, suggesting that endogenous ERRgamma plays an important role in osteoblast differentiation. In addition, ERRgamma significantly repressed Runx2 transactivity on osteocalcin and bone sialoprotein promoters. We also observed that ERRgamma physically interacts with Runx2 in vitro and in vivo and competes with p300 to repress Runx2 transactivity. Notably, intramuscular injection of ERRgamma strongly inhibited BMP2-induced ectopic bone formation in a dose-dependent manner. Taken together, these results suggest that ERRgamma is a novel negative regulator of osteoblast differentiation and bone formation via its regulation of Runx2 transactivity. PMID:19324883

  10. The Orphan Nuclear Receptor Estrogen Receptor-related Receptor γ Negatively Regulates BMP2-induced Osteoblast Differentiation and Bone Formation*

    PubMed Central

    Jeong, Byung-Chul; Lee, Yong-Soo; Park, Yun-Yong; Bae, In-Ho; Kim, Don-Kyu; Koo, Seung-Hoi; Choi, Hong-Ran; Kim, Sun-Hun; Franceschi, Renny T.; Koh, Jeong-Tae; Choi, Hueng-Sik

    2009-01-01

    Estrogen receptor-related receptor γ (ERRγ/ERR3/NR3B3) is a member of the orphan nuclear receptor with important functions in development and homeostasis. Recently it has been reported that ERRα is involved in osteoblast differentiation and bone formation. In the present study we examined the role of ERRγ in osteoblast differentiation. Here, we showed that ERRγ is expressed in osteoblast progenitors and primary osteoblasts, and its expression is increased temporarily by BMP2. Overexpression of ERRγ reduced BMP2-induced alkaline phosphatase activity and osteocalcin production as well as calcified nodule formation, whereas inhibition of ERRγ expression significantly enhanced BMP2-induced osteogenic differentiation and mineralization, suggesting that endogenous ERRγ plays an important role in osteoblast differentiation. In addition, ERRγ significantly repressed Runx2 transactivity on osteocalcin and bone sialoprotein promoters. We also observed that ERRγ physically interacts with Runx2 in vitro and in vivo and competes with p300 to repress Runx2 transactivity. Notably, intramuscular injection of ERRγ strongly inhibited BMP2-induced ectopic bone formation in a dose-dependent manner. Taken together, these results suggest that ERRγ is a novel negative regulator of osteoblast differentiation and bone formation via its regulation of Runx2 transactivity. PMID:19324883

  11. Ammonia Induces Autophagy through Dopamine Receptor D3 and MTOR

    PubMed Central

    Li, Zhiyuan; Ji, Xinmiao; Wang, Wenchao; Liu, Juanjuan; Liang, Xiaofei; Wu, Hong; Liu, Jing; Eggert, Ulrike S.; Liu, Qingsong

    2016-01-01

    Hyperammonemia is frequently seen in tumor microenvironments as well as in liver diseases where it can lead to severe brain damage or death. Ammonia induces autophagy, a mechanism that tumor cells may use to protect themselves from external stresses. However, how cells sense ammonia has been unclear. Here we show that culture medium alone containing Glutamine can generate milimolar of ammonia at 37 degrees in the absence of cells. In addition, we reveal that ammonia acts through the G protein-coupled receptor DRD3 (Dopamine receptor D3) to induce autophagy. At the same time, ammonia induces DRD3 degradation, which involves PIK3C3/VPS34-dependent pathways. Ammonia inhibits MTOR (mechanistic target of Rapamycin) activity and localization in cells, which is mediated by DRD3. Therefore, ammonia has dual roles in autophagy: one to induce autophagy through DRD3 and MTOR, the other to increase autophagosomal pH to inhibit autophagic flux. Our study not only adds a new sensing and output pathway for DRD3 that bridges ammonia sensing and autophagy induction, but also provides potential mechanisms for the clinical consequences of hyperammonemia in brain damage, neurodegenerative diseases and tumors. PMID:27077655

  12. The role of non-receptor protein tyrosine kinases in the excitotoxicity induced by the overactivation of NMDA receptors.

    PubMed

    Sun, Yongjun; Chen, You; Zhan, Liying; Zhang, Linan; Hu, Jie; Gao, Zibin

    2016-04-01

    Protein tyrosine phosphorylation is one of the primary modes of regulation of N-methyl-d-aspartate (NMDA) receptors. The non-receptor tyrosine kinases are one of the two types of protein tyrosine kinases that are involved in this process. The overactivation of NMDA receptors is a primary reason for neuron death following cerebral ischemia. Many studies have illustrated the important role of non-receptor tyrosine kinases in ischemia insults. This review introduces the roles of Src, Fyn, focal adhesion kinase, and proline-rich tyrosine kinase 2 in the excitotoxicity induced by the overactivation of NMDA receptors following cerebral ischemia. PMID:26540220

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

    SciTech Connect

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

    1991-06-01

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

  14. 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.

  15. ZINC-INDUCED EGF RECEPTOR SIGNALING REQUIRES SRC-MEDIATED PHOSPHORYLATION OF THE EGF RECEPTOR ON TYROSINE 845 (Y845)

    EPA Science Inventory

    ZINC-INDUCED EGF RECEPTOR SIGNALING REQUIRES Src-MEDIATED PHOSPHORYLATION OF THE EGF RECEPTOR ON TYROSINE 845 (Y845)
    Weidong Wu1, Lee M. Graves2, Gordon N. Gill3 and James M. Samet4 1Center for Environmental Medicine and Lung Biology; 2Department of Pharmacology, University o...

  16. Activation of type 5 metabotropic glutamate receptors attenuates deficits in cognitive flexibility induced by NMDA receptor blockade

    PubMed Central

    Stefani, Mark R.; Moghaddam, Bita

    2010-01-01

    Metabotropic glutamate (mGlu) receptors provide a mechanism by which the function of NMDA glutamate receptors can be modulated. As NMDA receptor hypofunction is implicated in the etiology of psychiatric disorders, including schizophrenia, the pharmacological regulation of mGlu receptor activity represents a promising therapeutic approach. We examined the effects of the positive allosteric mGlu5 receptor modulator 3- cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB), alone and in combination with the NMDA receptor antagonist MK-801, on a task measuring cognitive set-shifting ability. This task measures NMDA receptor-dependent cognitive abilities analogous to those impaired in schizophrenia. Systemic administration of CDPPB (10 & 30 mg/kg i.p) blocked MK-801 (0.1 mg/kg, i.p.)-induced impairments in set-shifting ability. The effect on learning was dose-dependent, with the 30 mg/kg dose having a greater effect than the 10 mg/kg dose across all trials. This ameliorative effect of CDPPB reflected a reduction in MK-801-induced perseverative responding. These results add to the evidence that mGlu5 receptors interact functionally with NMDA receptors to regulate behavior, and suggest that positive modulators of mGlu5 receptors may have therapeutic potential in the treatment of disorders, like schizophrenia, characterized by impairments in cognitive flexibility and memory. PMID:20371234

  17. Angiotensin II receptors and peritoneal dialysis-induced peritoneal fibrosis.

    PubMed

    Morinelli, Thomas A; Luttrell, Louis M; Strungs, Erik G; Ullian, Michael E

    2016-08-01

    The vasoactive hormone angiotensin II initiates its major hemodynamic effects through interaction with AT1 receptors, a member of the class of G protein-coupled receptors. Acting through its AT1R, angiotensin II regulates blood pressure and renal salt and water balance. Recent evidence points to additional pathological influences of activation of AT1R, in particular inflammation, fibrosis and atherosclerosis. The transcription factor nuclear factor κB, a key mediator in inflammation and atherosclerosis, can be activated by angiotensin II through a mechanism that may involve arrestin-dependent AT1 receptor internalization. Peritoneal dialysis is a therapeutic modality for treating patients with end-stage kidney disease. The effectiveness of peritoneal dialysis at removing waste from the circulation is compromised over time as a consequence of peritoneal dialysis-induced peritoneal fibrosis. The non-physiological dialysis solution used in peritoneal dialysis, i.e. highly concentrated, hyperosmotic glucose, acidic pH as well as large volumes infused into the peritoneal cavity, contributes to the development of fibrosis. Numerous trials have been conducted altering certain components of the peritoneal dialysis fluid in hopes of preventing or delaying the fibrotic response with limited success. We hypothesize that structural activation of AT1R by hyperosmotic peritoneal dialysis fluid activates the internalization process and subsequent signaling through the transcription factor nuclear factor κB, resulting in the generation of pro-fibrotic/pro-inflammatory mediators producing peritoneal fibrosis. PMID:27167177

  18. Carnosic acid inhibits TLR4-MyD88 signaling pathway in LPS-stimulated 3T3-L1 adipocytes

    PubMed Central

    Park, Mi-Young

    2014-01-01

    BACKGROUND/OBJECTIVES Carnosic acid (CA), found in rosemary (Rosemarinus officinalis) leaves, is known to exhibit anti-obesity and anti-inflammatory activities. However, whether its anti-inflammatory potency can contribute to the amelioration of obesity has not been elucidated. The aim of the current study was to investigate the effect of CA on Toll-like receptor 4 (TLR4) pathways in the presence of lipopolysaccharide (LPS) in 3T3-L1 adipocytes. MATERIALS/METHODS 3T3-L1 adipocytes were treated with CA (0-20 µM) for 1 h, followed by treatment with LPS for 30 min; mRNA expression of adipokines and protein expression of TLR4-related molecules were then measured. RESULTS LPS-stimulated 3T3-L1 adipocytes showed elevated mRNA expression of tumor necrosis factor (TNF)-α, interleukin-6, and monocyte chemoattractant protein-1, and CA significantly inhibited the expression of these adipokine genes. LPS-induced up regulation of TLR4, myeloid differentiation factor 88, TNF receptor-associated factor 6, and nuclear factor-κB, as well as phosphorylated extracellular receptor-activated kinase were also suppressed by pre-treatment of 3T3-L1 adipocytes with CA. CONCLUSIONS Results of this study suggest that CA directly inhibits TLR4-MyD88-dependent signaling pathways and decreases the inflammatory response in adipocytes. PMID:25324930

  19. Insulin decreases atherosclerosis by inducing endothelin receptor B expression

    PubMed Central

    Park, Kyoungmin; Mima, Akira; Li, Qian; Rask-Madsen, Christian; He, Pingnian; Mizutani, Koji; Katagiri, Sayaka; Maeda, Yasutaka; Wu, I-Hsien; Khamaisi, Mogher; Preil, Simone Rordam; Maddaloni, Ernesto; Sørensen, Ditte; Rasmussen, Lars Melholt; Huang, Paul L.; King, George L.

    2016-01-01

    Endothelial cell (EC) insulin resistance and dysfunction, caused by diabetes, accelerates atherosclerosis. It is unknown whether specifically enhancing EC-targeted insulin action can decrease atherosclerosis in diabetes. Accordingly, overexpressing insulin receptor substrate-1 (IRS1) in the endothelia of Apoe−/− mice (Irs1/Apoe−/−) increased insulin signaling and function in the aorta. Atherosclerosis was significantly reduced in Irs1/ApoE−/− mice on diet-induced hyperinsulinemia and hyperglycemia. The mechanism of insulin’s enhanced antiatherogenic actions in EC was related to remarkable induction of NO action, which increases endothelin receptor B (EDNRB) expression and intracellular [Ca2+]. Using the mice with knockin mutation of eNOS, which had Ser1176 mutated to alanine (AKI), deleting the only known mechanism for insulin to activate eNOS/NO pathway, we observed that IRS1 overexpression in the endothelia of Aki/ApoE−/− mice significantly decreased atherosclerosis. Interestingly, endothelial EDNRB expression was selectively reduced in intima of arteries from diabetic patients and rodents. However, endothelial EDNRB expression was upregulated by insulin via P13K/Akt pathway. Finally EDNRB deletion in EC of Ldlr−/− and Irs1/Ldlr−/− mice decreased NO production and accelerated atherosclerosis, compared with Ldlr−/− mice. Accelerated atherosclerosis in diabetes may be reduced by improving insulin signaling selectively via IRS1/Akt in the EC by inducing EDNRB expression and NO production. PMID:27200419

  20. Nicotinic acetylcholine receptors mediate donepezil-induced oligodendrocyte differentiation.

    PubMed

    Imamura, Osamu; Arai, Masaaki; Dateki, Minori; Ogata, Toru; Uchida, Ryuji; Tomoda, Hiroshi; Takishima, Kunio

    2015-12-01

    Oligodendrocytes are the myelin-forming cells of the central nervous system (CNS). Failure of myelin development and oligodendrocyte loss results in serious human disorders, including multiple sclerosis. Here, we show that donepezil, an acetlycholinesterase inhibitor developed for the treatment of Alzheimer's disease, can stimulate oligodendrocyte differentiation and maturation of neural stem cell-derived oligodendrocyte progenitor cells without affecting proliferation or cell viability. Transcripts for essential myelin-associated genes, such as PLP, MAG, MBP, CNPase, and MOG, in addition to transcription factors that regulate oligodendrocyte differentiation and myelination, were rapidly increased after treatment with donepezil. Furthermore, luciferase assays confirmed that both MAG and MBP promoters display increased activity upon donepezil-induced oligodendrocytes differentiation, suggesting that donepezil increases myelin gene expression mainly through enhanced transcription. We also found that the increase in the number of oligodendrocytes observed following donepezil treatment was significantly inhibited by the nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine, but not by the muscarinic acetylcholine receptor antagonist scopolamine. Moreover, donepezil-induced myelin-related gene expression was suppressed by mecamylamine at both the mRNA and protein level. These results suggest that donepezil stimulates oligodendrocyte differentiation and myelin-related gene expression via nAChRs in neural stem cell-derived oligodendrocyte progenitor cells. We show that donepezil, a drug for the treatment of Alzheimer disease, can stimulate oligodendrocyte differentiation and maturation of oligodendrocyte progenitor cells. Transcripts for essential myelin-associated genes, such as PLP, MAG, MBP, CNPase and MOG in addition to transcripton factors that regulate oligodendrocyte differentiation and myelination were rapidly increased after treatment with donepezil

  1. The metabolism of T4 and T3 in cultured chick-embryo heart cells.

    PubMed

    Dickstein, Y; Schwartz, H; Gross, J; Gordon, A

    1980-10-01

    Thyroxine (T4) and tri-iodothyronine (T3) were metabolized in cultured chick-embryo heart cells via inner-ring de-iodination and O-sulfation. The products of T3 metabolism were 3,3'T2, 3'T1, and sulfated esters of T3 and 3,3'T2. The major product of T4 degradation was 3,3',5'-T3 (rT3). ONo T3 was produced from T4. Propylthioracil inhibited the metabolism of T3. Pretreatment of cultures with T3 or T4 enhanced the metabolism of both hormones; actinomycin D and cycloheximide inhibited the stimulatory effect of T3. The stimulation by T3 was linearly related to the log of the concentration of T3 in cells grown in normal chick serum or in cells grown in dehormonized serum. These results suggest that thyroid hormones induced an increased synthesis of the enzymes involved in their metabolism and therefore may regulate their own disposal rate. PMID:7439522

  2. α-Melanocyte stimulating hormone attenuates dexamethasone-induced osteoblast damages through activating melanocortin receptor 4-SphK1 signaling.

    PubMed

    Guo, Shiguang; Xie, Yue; Fan, Jian-bo; Ji, Feng; Wang, Shouguo; Fei, Haodong

    2016-01-01

    Long-term glucocorticoid (GC) usage may cause non-traumatic femoral head osteonecrosis. Dexamethasone (Dex) is shown to exert potent cytotoxic effect to osteoblasts. Here, we investigated the potential activity of α-melanocyte stimulating hormone (α-MSH) against the process. Our data revealed that pretreatment of α-MSH significantly inhibited Dex-induced apoptosis and necrosis in both osteoblastic-like MC3T3-E1 cells and primary murine osteoblasts. Melanocortin receptor 4 (MC4R) acts as the receptor of α-MSH in mediating its actions in osteoblasts. The MC4R antagonist SHU9119, or shRNA-mediated knockdown of MC4R, almost abolished α-MSH-induced activation of downstream signalings (Akt and Erk1/2) and its pro-survival effect in osteoblasts. Further studies showed that α-MSH activated MC4R downstream sphingosine kinase 1 (SphK1) and increased cellular sphingosine-1-phosphate (S1P) content in MC3T3-E1 cells and primary murine osteoblasts, which were blocked by SHU9119 or MC4R shRNAs. SphK1 inhibition by the its inhibitor N,N-dimethylsphingosine (DMS), or SphK1 knockdown by targeted-shRNAs, largely attenuated α-MSH-mediated osteoblast protection against Dex. Together, these results suggest that α-MSH alleviates Dex-induced damages to cultured osteoblasts through activating MC4R-SphK1 signaling. PMID:26631960

  3. 5-HT2A receptor activation is necessary for CO2-induced arousal.

    PubMed

    Buchanan, Gordon F; Smith, Haleigh R; MacAskill, Amanda; Richerson, George B

    2015-07-01

    Hypercapnia-induced arousal from sleep is an important protective mechanism pertinent to a number of diseases. Most notably among these are the sudden infant death syndrome, obstructive sleep apnea and sudden unexpected death in epilepsy. Serotonin (5-HT) plays a significant role in hypercapnia-induced arousal. The mechanism of 5-HT's role in this protective response is unknown. Here we sought to identify the specific 5-HT receptor subtype(s) involved in this response. Wild-type mice were pretreated with antagonists against 5-HT receptor subtypes, as well as antagonists against adrenergic, cholinergic, histaminergic, dopaminergic, and orexinergic receptors before challenge with inspired CO2 or hypoxia. Antagonists of 5-HT(2A) receptors dose-dependently blocked CO2-induced arousal. The 5-HT(2C) receptor antagonist, RS-102221, and the 5-HT1A receptor agonist, 8-OH-DPAT, attenuated but did not completely block CO2-induced arousal. Blockade of non-5-HT receptors did not affect CO2-induced arousal. None of these drugs had any effect on hypoxia-induced arousal. 5-HT2 receptor agonists were given to mice in which 5-HT neurons had been genetically eliminated during embryonic life (Lmx1b(f/f/p)) and which are known to lack CO2-induced arousal. Application of agonists to 5-HT(2A), but not 5-HT(2C), receptors, dose-dependently restored CO2-induced arousal in these mice. These data identify the 5-HT(2A) receptor as an important mediator of CO2-induced arousal and suggest that, while 5-HT neurons can be independently activated to drive CO2-induced arousal, in the absence of 5-HT neurons and endogenous 5-HT, 5-HT receptor activation can act in a permissive fashion to facilitate CO2-induced arousal via another as yet unidentified chemosensor system. PMID:25925320

  4. 5-HT2A receptor activation is necessary for CO2-induced arousal

    PubMed Central

    Smith, Haleigh R.; MacAskill, Amanda; Richerson, George B.

    2015-01-01

    Hypercapnia-induced arousal from sleep is an important protective mechanism pertinent to a number of diseases. Most notably among these are the sudden infant death syndrome, obstructive sleep apnea and sudden unexpected death in epilepsy. Serotonin (5-HT) plays a significant role in hypercapnia-induced arousal. The mechanism of 5-HT's role in this protective response is unknown. Here we sought to identify the specific 5-HT receptor subtype(s) involved in this response. Wild-type mice were pretreated with antagonists against 5-HT receptor subtypes, as well as antagonists against adrenergic, cholinergic, histaminergic, dopaminergic, and orexinergic receptors before challenge with inspired CO2 or hypoxia. Antagonists of 5-HT2A receptors dose-dependently blocked CO2-induced arousal. The 5-HT2C receptor antagonist, RS-102221, and the 5-HT1A receptor agonist, 8-OH-DPAT, attenuated but did not completely block CO2-induced arousal. Blockade of non-5-HT receptors did not affect CO2-induced arousal. None of these drugs had any effect on hypoxia-induced arousal. 5-HT2 receptor agonists were given to mice in which 5-HT neurons had been genetically eliminated during embryonic life (Lmx1bf/f/p) and which are known to lack CO2-induced arousal. Application of agonists to 5-HT2A, but not 5-HT2C, receptors, dose-dependently restored CO2-induced arousal in these mice. These data identify the 5-HT2A receptor as an important mediator of CO2-induced arousal and suggest that, while 5-HT neurons can be independently activated to drive CO2-induced arousal, in the absence of 5-HT neurons and endogenous 5-HT, 5-HT receptor activation can act in a permissive fashion to facilitate CO2-induced arousal via another as yet unidentified chemosensor system. PMID:25925320

  5. Morphine-induced μ-Opioid Receptor Rapid Desensitization is independent of Receptor Phosphorylation and β-Arrestins

    PubMed Central

    Chu, Ji; Zheng, Hui; Loh, Horace H.; Law, Ping-Yee

    2008-01-01

    Receptor desensitization involving receptor phosphorylation and subsequent βArrestin (βArr) recruitment has been implicated in the tolerance development mediated by μ-opioid receptor (OPRM1). However, the roles of receptor phosphorylation and βArr on morphine-induced OPRM1 desensitization remain to be demonstrated. Using OPRM1-induced intracellular Ca2+ ([Ca2+]i )release to monitor receptor activation, as predicted, [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO), induced OPRM1 desensitization in a receptor phosphorylation- and βArr-dependent manner. The DAMGO-induced OPRM1 desensitization was attenuated significantly when phosphorylation deficient OPRM1 mutants or Mouse Embryonic Fibroblast (MEF) cells from βArr1 and 2 knockout mice were used in the studies. Specifically, DAMGO-induced desensitization was blunted in HEK293 cells expressing the OPRM1S375A mutant and was eliminated in MEF cells isolated from βArr2 knockout mice expressing the wild type OPRM1. However, although morphine also could induce a rapid desensitization on [Ca2+]i release to a greater extent than that of DAMGO and could induce the phosphorylation of Ser375 residue, morphine-induced desensitization was not influenced by mutating the phosphorylation sites or in MEF cells lacking βArr1 and 2. Hence, morphine could induce OPRM1 desensitization via pathway independent of βArr, thus suggesting the in vivo tolerance development to morphine can occur in the absence of βArr. PMID:18558479

  6. Ligand-induced IFN gamma receptor tyrosine phosphorylation couples the receptor to its signal transduction system (p91).

    PubMed Central

    Greenlund, A C; Farrar, M A; Viviano, B L; Schreiber, R D

    1994-01-01

    Herein we report that interferon-gamma (IFN gamma) induces the rapid and reversible tyrosine phosphorylation of the IFN gamma receptor. Using a panel of receptor intracellular domain mutants, we show that a membrane-proximal LPKS sequence (residues 266-269) is required for ligand-induced tyrosine kinase activation and/or kinase-receptor association and biological responsiveness, and a functionally critical membrane-distal tyrosine residue (Y440) is a target of the activated enzyme. The biological significance of Y440 phosphorylation was demonstrated by showing that a receptor-derived nonapeptide corresponding to receptor residues 436-444 and containing phosphorylated Y440 bound specifically to p91, blocked p91 phosphorylation and inhibited the generation of an active p91-containing transcription factor complex. In contrast, nonphosphorylated wild-type, phosphorylated mutant, or phosphorylated irrelevant peptides did not. Moreover, the phosphorylated Y440-containing peptide did not interact with a related but distinct latent transcription factor (p113) which is activatible by IFN alpha but not IFN gamma. These results thus document the specific and inducible association of p91 with the phosphorylated IFN gamma receptor and thereby elucidate the mechanism by which ligand couples the IFN gamma receptor to its signal transduction system. Images PMID:8156998

  7. Attenuation of D-1 antagonist-induced D-1 receptor upregulation by conccomitant D-2 receptor blockade

    SciTech Connect

    Parashos, S.A.; Barone, P.; Tucci, I.; Chase, T.N.

    1987-11-16

    The effect of chronic selective D-1 and/or D-2 dopamine receptor blockade on regional D-1 receptor binding was studied in rat brain following chronic treatment with the specific D-1 antagonist SCH 23390 and/or the predominantly D-2 antagonist haloperidol. D-1 receptor density and affinity were evaluated by quantitative autoradiography using /sup 125/I-SCH 23982. Chronic SCH 23390 treatment increased D-1 receptor density by 30 to 40% in the striatum, accumbens and tuberculum olfactorium; receptor affinity remained unchanged. Haloperidol had no effect on D-1 receptor Bmax or Kd values, although, when administered with SCH 23390, reduced the D-1 receptor upregulation induced by the D-1 antagonist in striatum and tuberculum olfactorium, but not in nucleus accumbens, These results may be attributable to D-1/D-2 dopamine receptor interactions occurring in the striatum and tuberculum olfactorium and may have implications for the prevention and treatment of drug-induced extrapyramidal disorders. 34 references, 1 figure, 2 tables.

  8. Chemotherapy-induced antitumor immunity requires formyl peptide receptor 1.

    PubMed

    Vacchelli, Erika; Ma, Yuting; Baracco, Elisa E; Sistigu, Antonella; Enot, David P; Pietrocola, Federico; Yang, Heng; Adjemian, Sandy; Chaba, Kariman; Semeraro, Michaela; Signore, Michele; De Ninno, Adele; Lucarini, Valeria; Peschiaroli, Francesca; Businaro, Luca; Gerardino, Annamaria; Manic, Gwenola; Ulas, Thomas; Günther, Patrick; Schultze, Joachim L; Kepp, Oliver; Stoll, Gautier; Lefebvre, Céline; Mulot, Claire; Castoldi, Francesca; Rusakiewicz, Sylvie; Ladoire, Sylvain; Apetoh, Lionel; Bravo-San Pedro, José Manuel; Lucattelli, Monica; Delarasse, Cécile; Boige, Valérie; Ducreux, Michel; Delaloge, Suzette; Borg, Christophe; André, Fabrice; Schiavoni, Giovanna; Vitale, Ilio; Laurent-Puig, Pierre; Mattei, Fabrizio; Zitvogel, Laurence; Kroemer, Guido

    2015-11-20

    Antitumor immunity driven by intratumoral dendritic cells contributes to the efficacy of anthracycline-based chemotherapy in cancer. We identified a loss-of-function allele of the gene coding for formyl peptide receptor 1 (FPR1) that was associated with poor metastasis-free and overall survival in breast and colorectal cancer patients receiving adjuvant chemotherapy. The therapeutic effects of anthracyclines were abrogated in tumor-bearing Fpr1(-/-) mice due to impaired antitumor immunity. Fpr1-deficient dendritic cells failed to approach dying cancer cells and, as a result, could not elicit antitumor T cell immunity. Experiments performed in a microfluidic device confirmed that FPR1 and its ligand, annexin-1, promoted stable interactions between dying cancer cells and human or murine leukocytes. Altogether, these results highlight the importance of FPR1 in chemotherapy-induced anticancer immune responses. PMID:26516201

  9. T3DB: the toxic exposome database

    PubMed Central

    Wishart, David; Arndt, David; Pon, Allison; Sajed, Tanvir; Guo, An Chi; Djoumbou, Yannick; Knox, Craig; Wilson, Michael; Liang, Yongjie; Grant, Jason; Liu, Yifeng; Goldansaz, Seyed Ali; Rappaport, Stephen M.

    2015-01-01

    The exposome is defined as the totality of all human environmental exposures from conception to death. It is often regarded as the complement to the genome, with the interaction between the exposome and the genome ultimately determining one's phenotype. The ‘toxic exposome’ is the complete collection of chronically or acutely toxic compounds to which humans can be exposed. Considerable interest in defining the toxic exposome has been spurred on by the realization that most human injuries, deaths and diseases are directly or indirectly caused by toxic substances found in the air, water, food, home or workplace. The Toxin-Toxin-Target Database (T3DB - www.t3db.ca) is a resource that was specifically designed to capture information about the toxic exposome. Originally released in 2010, the first version of T3DB contained data on nearly 2900 common toxic substances along with detailed information on their chemical properties, descriptions, targets, toxic effects, toxicity thresholds, sequences (for both targets and toxins), mechanisms and references. To more closely align itself with the needs of epidemiologists, toxicologists and exposome scientists, the latest release of T3DB has been substantially upgraded to include many more compounds (>3600), targets (>2000) and gene expression datasets (>15 000 genes). It now includes extensive data on ‘normal’ toxic compound concentrations in human biofluids as well as detailed chemical taxonomies, informative chemical ontologies and a large number of referential NMR, MS/MS and GC-MS spectra. This manuscript describes the most recent update to the T3DB, which was previously featured in the 2010 NAR Database Issue. PMID:25378312

  10. Proteasome involvement in agonist-induced down-regulation of mu and delta opioid receptors.

    PubMed

    Chaturvedi, K; Bandari, P; Chinen, N; Howells, R D

    2001-04-13

    This study investigated the mechanism of agonist-induced opioid receptor down-regulation. Incubation of HEK 293 cells expressing FLAG-tagged delta and mu receptors with agonists caused a time-dependent decrease in opioid receptor levels assayed by immunoblotting. Pulse-chase experiments using [(35)S]methionine metabolic labeling indicated that the turnover rate of delta receptors was accelerated 5-fold following agonist stimulation. Inactivation of functional G(i) and G(o) proteins by pertussis toxin-attenuated down-regulation of the mu opioid receptor, while down-regulation of the delta opioid receptor was unaffected. Pretreatment of cells with inhibitors of lysosomal proteases, calpain, and caspases had little effect on mu and delta opioid receptor down-regulation. In marked contrast, pretreatment with proteasome inhibitors attenuated agonist-induced mu and delta receptor down-regulation. In addition, incubation of cells with proteasome inhibitors in the absence of agonists increased steady-state mu and delta opioid receptor levels. Immunoprecipitation of mu and delta opioid receptors followed by immunoblotting with ubiquitin antibodies suggested that preincubation with proteasome inhibitors promoted accumulation of polyubiquitinated receptors. These data provide evidence that the ubiquitin/proteasome pathway plays a role in agonist-induced down-regulation and basal turnover of opioid receptors. PMID:11152677

  11. Kainate receptor activation induces glycine receptor endocytosis through PKC deSUMOylation.

    PubMed

    Sun, Hao; Lu, Li; Zuo, Yong; Wang, Yan; Jiao, Yingfu; Zeng, Wei-Zheng; Huang, Chao; Zhu, Michael X; Zamponi, Gerald W; Zhou, Tong; Xu, Tian-Le; Cheng, Jinke; Li, Yong

    2014-01-01

    Surface expression and regulated endocytosis of glycine receptors (GlyRs) play a critical function in balancing neuronal excitability. SUMOylation (SUMO modification) is of critical importance for maintaining neuronal function in the central nervous system. Here we show that activation of kainate receptors (KARs) causes GlyR endocytosis in a calcium- and protein kinase C (PKC)-dependent manner, leading to reduced GlyR-mediated synaptic activity in cultured spinal cord neurons and the superficial dorsal horn of rat spinal cord slices. This effect requires SUMO1/sentrin-specific peptidase 1 (SENP1)-mediated deSUMOylation of PKC, indicating that the crosstalk between KARs and GlyRs relies on the SUMOylation status of PKC. SENP1-mediated deSUMOylation of PKC is involved in the kainate-induced GlyR endocytosis and thus plays an important role in the anti-homeostatic regulation between excitatory and inhibitory ligand-gated ion channels. Altogether, we have identified a SUMOylation-dependent regulatory pathway for GlyR endocytosis, which may have important physiological implications for proper neuronal excitability. PMID:25236484

  12. Interaction of myocardial insulin receptor and IGF receptor signaling in exercise-induced cardiac hypertrophy

    PubMed Central

    Ikeda, Hiroyuki; Shiojima, Ichiro; Ozasa, Yukako; Yoshida, Masashi; Holzenberger, Martin; Kahn, C Ronald; Walsh, Kenneth; Igarashi, Takashi; Abel, E Dale; Komuro, Issei

    2016-01-01

    Insulin-like growth factor-1 (IGF-1) signaling has recently been implicated in the development of cardiac hypertrophy after long-term endurance training, via mechanisms that may involve energetic stress. Given the potential overlap of insulin and IGF-1 signaling we sought to determine if both signaling pathways could contribute to exercise-induced cardiac hypertrophy following shorter-term exercise training. Studies were performed in mice with cardiac-specific IGF-1 receptor (IGF1R) knockout (CIGFRKO), mice with cardiac-specific insulin receptor (IR) knockout (CIRKO), CIGFRKO mice that lacked one IR allele in cardiomyocytes (IGFR−/−IR+/−), and CIRKO mice that lacked one IGF1R allele in cardiomyocytes (IGFR+/−IR−/−). Intravenous administration of IGF-1 or 75 hours of swimming over 4 weeks increased IGF1R tyrosine phosphorylation in the heart in control and CIRKO mice but not in CIGFRKO mice. Intriguingly, IR tyrosine phosphorylation in the heart was also increased following IGF-1 administration or exercise training in control and CIGFRKO mice but not in CIRKO mice. The extent of cardiac hypertrophy following exercise training in CIGFRKO and CIRKO mice was comparable to that in control mice. In contrast, exercise-induced cardiac hypertrophy was significantly attenuated in IGFR−/−IR+/− and IGFR+/−IR−/− mice. Thus, IGF-1 and exercise activates both IGF1R and IR in the heart, and IGF1R- and IR-mediated signals may serve redundant roles in the hypertrophic responses of the heart to exercise training. PMID:19744489

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

    PubMed

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

    2013-11-01

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

  14. Bisphenol A Diglycidyl Ether Induces Adipogenic Differentiation of Multipotent Stromal Stem Cells through a Peroxisome Proliferator–Activated Receptor Gamma-Independent Mechanism

    PubMed Central

    Chamorro-García, Raquel; Kirchner, Séverine; Li, Xia; Janesick, Amanda; Casey, Stephanie C.; Chow, Connie

    2012-01-01

    Background: Bisphenol A (BPA) and bisphenol A diglycidyl ether (BADGE), used in manufacturing coatings and resins, leach from packaging materials into food. Numerous studies suggested that BPA and BADGE may have adverse effects on human health, including the possibility that exposure to such chemicals can be superimposed on traditional risk factors to initiate or exacerbate the development of obesity. BPA is a suspected obesogen, whereas BADGE, described as a peroxisome proliferator–activated receptor gamma (PPARγ) antagonist, could reduce weight gain. Objectives: We sought to test the adipogenic effects of BADGE in a biologically relevant cell culture model. Methods: We used multipotent mesenchymal stromal stem cells (MSCs) to study the adipogenic capacity of BADGE and BPA and evaluated their effects on adipogenesis, osteogenesis, gene expression, and nuclear receptor activation. Discussion: BADGE induced adipogenesis in human and mouse MSCs, as well as in mouse 3T3-L1 preadipocytes. In contrast, BPA failed to promote adipogenesis in MSCs, but induced adipogenesis in 3T3-L1 cells. BADGE exposure elicited an adipogenic gene expression profile, and its ability to induce adipogenesis and the expression of adipogenic genes was not blocked by known PPARγ antagonists. Neither BADGE nor BPA activated or antagonized retinoid “X” receptor (RXR) or PPARγ in transient transfection assays. Conclusions: BADGE can induce adipogenic differentiation in both MSCs and in preadipocytes at low nanomolar concentrations comparable to those that have been observed in limited human biomonitoring. BADGE probably acts through a mechanism that is downstream of, or parallel to, PPARγ. PMID:22763116

  15. Development of a peptide-based inducer of nuclear receptors degradation.

    PubMed

    Demizu, Yosuke; Ohoka, Nobumichi; Nagakubo, Takaya; Yamashita, Hiroko; Misawa, Takashi; Okuhira, Keiichiro; Naito, Mikihiko; Kurihara, Masaaki

    2016-06-01

    A peptide-based protein knockdown system for inducing nuclear receptors degradation via the ubiquitin-proteasome system was developed. Specifically, the designed molecules were composed of two biologically active scaffolds: a peptide that binds to the estrogen receptor α (ERα) surface and an MV1 molecule that binds to cellular inhibitors of apoptosis proteins (IAP: cIAP1/cIAP2/XIAP) to induce ubiquitylation of the ERα. The hybrid peptides induced IAP-mediated ubiquitylation followed by proteasomal degradation of the ERα. Those peptides were also applicable for inducing androgen receptor (AR) degradation. PMID:27086122

  16. Activation of retinoid X receptors induces apoptosis in HL-60 cell lines.

    PubMed Central

    Nagy, L; Thomázy, V A; Shipley, G L; Fésüs, L; Lamph, W; Heyman, R A; Chandraratna, R A; Davies, P J

    1995-01-01

    Retinoids induce myeloblastic leukemia (HL-60) cells to differentiate into granulocytes, which subsequently die by apoptosis. Retinoid action is mediated through at least two classes of nuclear receptors: retinoic acid receptors, which bind both all-trans retinoic acid and 9-cis retinoic acid, and retinoid X receptors, which bind only 9-cis retinoic acid. Using receptor-selective synthetic retinoids and HL-60 cell sublines with different retinoid responsiveness, we have investigated the contribution that each class of receptors makes to the processes of cellular differentiation and death. Our results demonstrate that ligand activation of retinoic acid receptors is sufficient to induce differentiation, whereas ligand activation of retinoid X receptors is essential for the induction of apoptosis in HL-60 cell lines. PMID:7791761

  17. Involvement of histamine H4 and H1 receptors in scratching induced by histamine receptor agonists in Balb C mice.

    PubMed

    Bell, J K; McQueen, D S; Rees, J L

    2004-05-01

    The role of histamine H(1), H(2), H(3) and H(4) receptors in acute itch induced by histamine was investigated in female BalbC mice. Scratching was induced by intradermal injections of pruritogen into the back of the neck and "itch" assessed by quantifying the scratching evoked. Histamine (0.03-80 micromol), histamine-trifluoromethyl-toluidine (HTMT, H(1) agonist, 0.002-2 micromol), clobenpropit (H(4) agonist, H(3) antagonist, 0.002-0.6 micromol) and to a lesser extent imetit (H(3)/H(4) agonist, 0.03-3 micromol) all induced dose-dependent scratching. Dimaprit (H(2) agonist, 0.04-40 micromol) did not cause scratching. Mepyramine (H(1) antagonist, 20 mg kg(-1), i.p.) reduced scratching evoked by histamine and HTMT, but not that caused by H(3) or H(4) agonists. Thioperamide (H(3)/H(4) antagonist, 20 mg kg(-1), i.p.) reduced scratching induced by histamine, H(3) and H(4) agonists, but not that caused by HTMT. The non-sedating H(1) antagonist, terfenadine, also significantly reduced the scratching induced by the H(1) agonist, HTMT. Cimetidine (H(2) antagonist, 20 mg kg(-1), i.p.) did not affect histamine-induced scratching. These results indicate that activation of histamine H(4) receptors causes itch in mice, in addition to the previously recognised role for H(1) receptors in evoking itch. Histamine H(4) receptor antagonists therefore merit investigation as antipruritic agents. PMID:15066908

  18. Signaling Mechanism of Cannabinoid Receptor-2 Activation-Induced β-Endorphin Release.

    PubMed

    Gao, Fang; Zhang, Ling-Hong; Su, Tang-Feng; Li, Lin; Zhou, Rui; Peng, Miao; Wu, Cai-Hua; Yuan, Xiao-Cui; Sun, Ning; Meng, Xian-Fang; Tian, Bo; Shi, Jing; Pan, Hui-Lin; Li, Man

    2016-08-01

    Activation of cannabinoid receptor-2 (CB2) results in β-endorphin release from keratinocytes, which then acts on primary afferent neurons to inhibit nociception. However, the underlying mechanism is still unknown. The CB2 receptor is generally thought to couple to Gi/o to inhibit cAMP production, which cannot explain the peripheral stimulatory effects of CB2 receptor activation. In this study, we found that in a keratinocyte cell line, the Gβγ subunits from Gi/o, but not Gαs, were involved in CB2 receptor activation-induced β-endorphin release. Inhibition of MAPK kinase, but not PLC, abolished CB2 receptor activation-induced β-endorphin release. Also, CB2 receptor activation significantly increased intracellular Ca(2+). Treatment with BAPTA-AM or thapsigargin blocked CB2 receptor activation-induced β-endorphin release. Using a rat model of inflammatory pain, we showed that the MAPK kinase inhibitor PD98059 abolished the peripheral effect of the CB2 receptor agonist on nociception. We thus present a novel mechanism of CB2 receptor activation-induced β-endorphin release through Gi/o-Gβγ-MAPK-Ca(2+) signaling pathway. Our data also suggest that stimulation of MAPK contributes to the peripheral analgesic effect of CB2 receptor agonists. PMID:26108183

  19. Blockade of the N-Methyl-D-Aspartate Glutamate Receptor Ameliorates Lipopolysaccharide-Induced Renal Insufficiency

    PubMed Central

    Huang, Ho-Shiang; Ma, Ming-Chieh

    2015-01-01

    N-methyl-D-aspartate (NMDA) receptor activation in rat kidney reduces renal perfusion and ultrafiltration. Hypoperfusion-induced ischemia is the most frequent cause of functional insufficiency in the endotoxemic kidney. Here, we used non-hypotensive rat model of lipopolysaccharide-induced endotoxemia to examine whether NMDA receptor hyperfunction contributes to acute kidney injury. Lipopolysaccharide-induced renal damage via increased enzymuria and hemodynamic impairments were ameliorated by co-treatment with the NMDA receptor blocker, MK-801. The NMDA receptor NR1 subunit in the rat kidney mainly co-localized with serine racemase, an enzyme responsible for synthesizing the NMDA receptor co-agonist, D-serine. The NMDA receptor hyperfunction in lipopolysaccharide-treated kidneys was demonstrated by NR1 and serine racemase upregulation, particularly in renal tubules, and by increased D-serine levels. Lipopolysaccharide also induced cell damage in cultured tubular cell lines and primary rat proximal tubular cells. This damage was mitigated by MK-801 and by small interfering RNA targeting NR1. Lipopolysaccharide increased cytokine release in tubular cell lines via toll-like receptor 4. The release of interleukin-1β from these cells are the most abundant. An interleukin-1 receptor antagonist not only attenuated cell death but also abolished lipopolysaccharide-induced NR1 and serine racemase upregulation and increases in D-serine secretion, suggesting that interleukin-1β-mediated NMDA receptor hyperfunction participates in lipopolysaccharide-induced tubular damage. The results of this study indicate NMDA receptor hyperfunction via cytokine effect participates in lipopolysaccharide-induced renal insufficiency. Blockade of NMDA receptors may represent a promising therapeutic strategy for the treatment of sepsis-associated renal failure. PMID:26133372

  20. Thyroid active agents T3 and PTU differentially affect immune gene transcripts in the head kidney of rainbow trout (Oncorynchus mykiss).

    PubMed

    Quesada-García, Alba; Encinas, Paloma; Valdehita, Ana; Baumann, Lisa; Segner, Helmut; Coll, Julio M; Navas, José M

    2016-05-01

    In mammals, numerous reports describe an immunomodulating effect of thyroid-active compounds. In contrast, only few reports have been published on this subject in fish. We previously demonstrated that immune cells of rainbow trout (Oncorhynchus mykiss) possess thyroid hormone receptors (THRs) and that exposure of trout to the thyroid hormone 3,3',5-triiodo-l-thyronine (T3) or the antithyroid drug propylthiouracil (PTU) alters immune cell transcript levels of THR and several immune genes. The present study aims to further characterize the immunomodulating action of thyroid-active compounds in trout immune cells. We report here the use of a custom-designed 60-mer oligo immune-targeted microarray for rainbow trout to analyze the gene expression profiles induced in the head kidney by T3 and PTU. Morphometric analyses of the thyroid showed that PTU exposure increased the size of the epithelial cells, whereas T3 induced no significant effects. Both T3 and PTU had diverse and partly contrasting effects on immune transcript profiles. The strongest differential effects of T3 and PTU on gene expressions were those targeting the Mitogen Associated Protein Kinase (MAPK), NFkB, Natural Killer (NK) and Toll-Like Receptor (TLR) pathways, a number of multipath genes (MPG) such as those encoding pleiotropic transcription factors (atf1, junb, myc), as well as important pro-inflammatory genes (tnfa, tnf6, il1b) and interferon-related genes (ifng, irf10). With these results we show for the first time in a fish species that the in vivo thyroidal status modulates a diversity of immune genes and pathways. This knowledge provides the basis to investigate both mechanisms and consequences of thyroid hormone- and thyroid disruptor-mediated immunomodulation for the immunocompetence of fish. PMID:26963519

  1. Cyclic mismatch binding ligand CMBL4 binds to the 5′-T-3′/5′-GG-3′ site by inducing the flipping out of thymine base

    PubMed Central

    Mukherjee, Sanjukta; Dohno, Chikara; Asano, Kaori; Nakatani, Kazuhiko

    2016-01-01

    A newly designed cyclic bis-naphthyridine carbamate dimer CMBL4 with a limited conformational flexibility was synthesized and characterized. Absorption spectra revealed that two naphthyridines in CMBL4 were stacked on each other in aqueous solutions. The most efficient binding of CMBL4 to DNA was observed for the sequence 5′-T-3′/5′-GG-3′ (T/GG) with the formation of a 1:1 complex, which is one of possible structural elements involved in the higher order structures of (TGG)n repeat DNA triggering the genome microdeletion. Surface plasmon resonance assay also showed the binding of CMBL4 with TGG repeat DNA. Potassium permanganate oxidation studies of CMBL4-bound duplex containing the T/GG site showed that the CMBL4-binding accelerated the oxidation of thymine at that site, which suggests the flipping out of the thymine base from a π-stack. Preferential binding was observed for CMBL4 compared with its acyclic variants, which suggests the marked significance of the macrocyclic structure for the recognition of the T/GG site. PMID:27466390

  2. A Herbal Formula HT048, Citrus unshiu and Crataegus pinnatifida, Prevents Obesity by Inhibiting Adipogenesis and Lipogenesis in 3T3-L1 Preadipocytes and HFD-Induced Obese Rats.

    PubMed

    Lee, Yoon Hee; Kim, Young-Sik; Song, Mikyung; Lee, Minsu; Park, Juyeon; Kim, Hocheol

    2015-01-01

    HT048 is a combination composed of Crataegus pinnatifida leaf and Citrus unshiu peel extracts. This study aimed to investigate potential anti-obesity effect of the combination. The 3T3-L1 adipocytes were treated with different doses of HT048 and triglyceride accumulation, glycerol release and adipogenesis-related genes were analyzed. For in vivo study, male Sprague Dawley rats were divided according to experimental diets: the chow diet group, the high-fat diet (HFD) group, the HFD supplemented with orlistat group, the HFD supplemented with HT048 group (0.2% or 0.4%) for 12 weeks. We measured the body weight, serum lipid levels and the expression of genes involved lipid metabolism. HT048 treatment dose-dependently suppressed adipocyte differentiation and stimulated glycerol release. The expressions of PPARγ and C/EBPα mRNA were decreased by HT048 treatment in adipocytes. HT048 supplementation significantly reduced the body and fat weights in vivo. Serum lipid levels were significantly lower in the HT048 supplemented groups than those of the HFD group. Expression of the hepatic lipogenesis-related genes were decreased and expression of the β-oxidation-related genes were increased in rats fed HT048 compared to that of animals fed HFD. These results suggest that HT048 has a potential benefit in preventing obesity through the inhibition of lipogenesis and adipogenesis. PMID:26016552

  3. Drug-induced up-regulation of dopamine D2 receptors on cultured cells.

    PubMed

    Starr, S; Kozell, L B; Neve, K A

    1995-08-01

    Ligand-induced up-regulation of recombinant dopamine D2 receptors was assessed using C6 glioma cells stably expressing the short (415-amino-acid; D2s) and long (444-amino-acid; D2L) forms of the receptor. Overnight treatment of C6-D2L cells with N-propylnorapomorphine (NPA) caused a time- and concentration-dependent increase in the density of receptors, as assessed by the binding of radioligand to membranes prepared from the cells, with no change in the affinity of the receptors for the radioligand. The effect of 10 microM NPA was maximal after 10 h, at which time the density of D2L receptors was more than doubled. The agonists dopamine and quinpirole also increased the density of D2L receptors. The receptor up-regulation was not specific for agonists, because the antagonists epidepride, sulpiride, and domperidone caused smaller (30-60%) increases in receptor density. Prolonged treatment with 10 microM NPA desensitized D2L receptors, as evidenced by a reduced ability of dopamine to inhibit adenylyl cyclase, whereas treatment with sulpiride was associated with an enhanced responsiveness to dopamine. The magnitude of NPA-induced receptor up-regulation in each of four clonal lines of C6-D2L cells (mean increase, 80%) was greater than in all four lines of C6-D2S cells (33%). Inactivation of pertussis toxin-sensitive G proteins had no effect on the basal density of D2L receptors or on the NPA-induced receptor up-regulation. Treatment with 5 micrograms/ml of cycloheximide, on the other hand, decreased the basal density of receptors and attenuated, but did not prevent, the NPA-induced increase.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7616211

  4. 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

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

    PubMed

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

    2015-11-11

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

  6. 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

    2016-01-01

    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

  7. Hyperthermia restores apoptosis induced by death receptors through aggregation-induced c-FLIP cytosolic depletion.

    PubMed

    Morlé, A; Garrido, C; Micheau, O

    2015-01-01

    TRAIL is involved in immune tumor surveillance and is considered a promising anti-cancer agent owing to its limited side effects on healthy cells. However, some cancer cells display resistance, or become resistant to TRAIL-induced cell death. Hyperthermia can enhance sensitivity to TRAIL-induced cell death in various resistant cancer cell lines, including lung, breast, colon or prostate carcinomas. Mild heat shock treatment has been proposed to restore Fas ligand or TRAIL-induced apoptosis through c-FLIP degradation or the mitochondrial pathway. We demonstrate here that neither the mitochondria nor c-FLIP degradation are required for TRAIL-induced cell death restoration during hyperthermia. Our data provide evidence that insolubilization of c-FLIP, alone, is sufficient to enhance apoptosis induced by death receptors. Hyperthermia induced c-FLIP depletion from the cytosolic fraction, without apparent degradation, thereby preventing c-FLIP recruitment to the TRAIL DISC and allowing efficient caspase-8 cleavage and apoptosis. Hyperthermia-induced c-FLIP depletion was independent of c-FLIP DED2 FL chain assembly motif or ubiquitination-mediated c-FLIP degradation, as assessed using c-FLIP point mutants on lysine 167 and 195 or threonine 166, a phosphorylation site known to regulate ubiquitination of c-FLIP. Rather, c-FLIP depletion was associated with aggregation, because addition of glycerol not only prevented the loss of c-FLIP from the cytosol but also enabled c-FLIP recruitment within the TRAIL DISC, thus inhibiting TRAIL-induced apoptosis during hyperthermia. Altogether our results demonstrate that c-FLIP is a thermosensitive protein whose targeting by hyperthermia allows restoration of apoptosis induced by TNF ligands, including TRAIL. Our findings suggest that combining TRAIL agonists with whole-body or localized hyperthermia may be an interesting approach in cancer therapy. PMID:25675293

  8. Inhibition of prostaglandin E2 receptor EP3 mitigates thrombin-induced brain injury.

    PubMed

    Han, Xiaoning; Lan, Xi; Li, Qiang; Gao, Yufeng; Zhu, Wei; Cheng, Tian; Maruyama, Takayuki; Wang, Jian

    2016-06-01

    Prostaglandin E2 EP3 receptor is the only prostaglandin E2 receptor that couples to multiple G-proteins, but its role in thrombin-induced brain injury is unclear. In the present study, we exposed mouse hippocampal slice cultures to thrombin in vitro and injected mice with intrastriatal thrombin in vivo to investigate the role of EP3 receptor in thrombin-induced brain injury and explore its underlying cellular and molecular mechanisms. In vitro, EP3 receptor inhibition reduced thrombin-induced hippocampal CA1 cell death. In vivo, EP3 receptor was expressed in astrocytes and microglia in the perilesional region. EP3 receptor inhibition reduced lesion volume, neurologic deficit, cell death, matrix metalloproteinase-9 activity, neutrophil infiltration, and the number of CD68(+) microglia, but increased the number of Ym-1(+) M2 microglia. RhoA-Rho kinase levels were increased after thrombin injection and were decreased by EP3 receptor inhibition. In mice that received an intrastriatal injection of autologous arterial blood, inhibition of thrombin activity with hirudin decreased RhoA expression compared with that in vehicle-treated mice. However, EP3 receptor activation reversed this effect of hirudin. These findings show that prostaglandin E2 EP3 receptor contributes to thrombin-induced brain damage via Rho-Rho kinase-mediated cytotoxicity and proinflammatory responses. PMID:26661165

  9. Acetylcholine receptor-inducing factor from chicken brain increases the level of mRNA encoding the receptor. alpha. subunit

    SciTech Connect

    Harris, D.A.; Falls, D.L.; Dill-Devor, R.M.; Fischbach, G.D. )

    1988-03-01

    A 42-kDa glycoprotein isolated from chicken brain, referred to as acetylcholine receptor-inducing activity (ARIA), that stimulates the rate of incorporation of acetylcholine receptors into the surface of chicken myotubes may play a role in the nerve-induced accumulation of receptors at developing neuromuscular synapses. Using nuclease-protection assays, the authors have found that ARIA causes a 2- to 16-fold increase in the level of mRNA encoding the {alpha} subunit of the receptor, with little or no change in the levels of {gamma}- and {delta}-subunit messengers. ARIA also increases the amount of a putative nuclear precursor of {alpha}-subunit mRNA, consistent with an activation of gene transcription. These results suggest that the concentration of {alpha} subunit may limit the rate of biosynthesis of the acetylcholine receptors in chicken myotubes. They also indicate that neuronal factors can regulate the expression of receptor subunit genes in a selective manner. Tetrodotoxin, 8-bromo-cAMP, and forskolin also increase the amount of {alpha}-subunit mRNA, with little change in the amount of {gamma}- and {delta}-subunit mRNAs. Unlike ARIA, however, these agents have little effect on the concentration of the {alpha}-subunit nuclear precursor.

  10. Monocyte chemoattractant protein-1-induced CCR2B receptor desensitization mediated by the G protein-coupled receptor kinase 2

    PubMed Central

    Aragay, A. M.; Mellado, M.; Frade, J. M. R.; Martin, A. M.; Jimenez-Sainz, M. C.; Martinez-A, C.; Mayor, F.

    1998-01-01

    Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine cytokine family, whose physiological function is mediated by binding to the CCR2 and CCR4 receptors, which are members of the G protein-coupled receptor family. MCP-1 plays a critical role in both activation and migration of leukocytes. Rapid chemokine receptor desensitization is very likely essential for accurate chemotaxis. In this report, we show that MCP-1 binding to the CCR2 receptor in Mono Mac 1 cells promotes the rapid desensitization of MCP-1-induced calcium flux responses. This desensitization correlates with the Ser/Thr phosphorylation of the receptor and with the transient translocation of the G protein-coupled receptor kinase 2 (GRK2, also called β-adrenergic kinase 1 or βARK1) to the membrane. We also demonstrate that GRK2 and the uncoupling protein β-arrestin associate with the receptor, forming a macromolecular complex shortly after MCP-1 binding. Calcium flux responses to MCP-1 in HEK293 cells expressing the CCR2B receptor were also markedly reduced upon cotransfection with GRK2 or the homologous kinase GRK3. Nevertheless, expression of the GRK2 dominant-negative mutant βARK-K220R did not affect the initial calcium response, but favored receptor response to a subsequent challenge by agonists. The modulation of the CCR2B receptor by GRK2 suggests an important role for this kinase in the regulation of monocyte and lymphocyte response to chemokines. PMID:9501202

  11. Alterations in insulin binding accompanying differentiation of 3T3-L1 preadipocytes.

    PubMed Central

    Reed, B C; Kaufmann, S H; Mackall, J C; Student, A K; Lane, M D

    1977-01-01

    Expression of the adipocyte phenotype by differentiating 3T3-L1 preadipocytes occurs upon exposure of the cells to insulin. Differentiation-linked changes in 125I-labeled insulin binding to 3T3-L1 cells were monitored and compared with those in nondifferentiating 3T3-C2 controls treated similarly. Without chronic insulin treatment, 3T3-L1 cells failed to express the adipocyte phenotype but maintained a level of 25,000-35,000 insulin-binding sites per cell. Treatment of 3T3-L1 cells with insulin resulted in an initial suppression of insulin binding followed by a 12-fold increase that paralleled the appearance of differentiated cells. A maximum of 170,000 insulin-binding sites per cell was attained for a population in which greater than 75% of the cells had differentiated. The increase of insulin receptor level appears to be differentiation-dependent and is not a general response of cells to the culture conditions. 3T3-C2 cells maintained in the presence of insulin for 30 days exhibited the undifferentiated phenotype and suppressed levels of insulin binding (35,000 sites per cell). The binding capacity of 3T3-L1 cells for epidermal growth factor remained unchanged between 25,000 and 40;000 sites per cell and was independent of the state of differentiation. Thus, induction by insulin in receptor-specific changes. Insulin receptors increase in number but epidermal growth factor receptors remain constant. PMID:303773

  12. Pregnane X receptor and drug-induced liver injury

    PubMed Central

    Wang, Yue-Ming; Chai, Sergio C.; Brewer, Christopher T; Chen, Taosheng

    2014-01-01

    Introduction The liver plays a central role in transforming and clearing foreign substances. The continuous exposure of the liver to xenobiotics sometimes leads to impaired liver function, referred to as drug-induced liver injury (DILI). The pregnane X receptor (PXR) tightly regulates the expression of genes in the hepatic drug-clearance system and its undesired activation plays a role in DILI. Areas covered This review focuses on the recent progress in understanding PXR-mediated DILI and highlights the efforts made to assess and manage PXR-mediated DILI during drug development. Expert opinion Future efforts are needed to further elucidate the mechanisms of PXR-mediated liver injury, including the epigenetic regulation and polymorphisms of PXR. Novel in vitro models containing functional PXR could improve our ability to predict and assess DILI during drug development. PXR inhibitors may provide chemical tools to validate the potential of PXR as a therapetic target and to develop drugs to be used in the clinic to manage PXR-mediated DILI. PMID:25252616

  13. Differential subcellular distribution of rat brain dopamine receptors and subtype-specific redistribution induced by cocaine

    PubMed Central

    Voulalas, Pamela J.; Schetz, John; Undieh, Ashiwel S.

    2011-01-01

    We investigated the subcellular distribution of dopamine D1, D2 and D5 receptor subtypes in rat frontal cortex, and examined whether psychostimulant-induced elevation of synaptic dopamine could alter the receptor distribution. Differential detergent solubilization and density gradient centrifugation were used to separate various subcellular fractions, followed by semi-quantitative determination of the relative abundance of specific receptor proteins in each fraction. D1 receptors were predominantly localized to detergent-resistant membranes, and a portion of these receptors also floated on sucrose gradients. These properties are characteristic of proteins found in lipid rafts and caveolae. D2 receptors exhibited variable distribution between cytoplasmic, detergent-soluble and detergent-resistant membrane fractions, yet were not present in buoyant membranes. Most D5 receptor immunoreactivity was distributed into the cytoplasmic fraction, failing to sediment at forces up to 300,000g, while the remainder was localized to detergent-soluble membranes in cortex. D5 receptors were undetectable in detergent-resistant fractions or raft-like subdomains. Following daily cocaine administration for seven days, a significant portion of D1 receptors translocated from detergent-resistant membranes to detergent-soluble membranes and the cytoplasmic fraction. The distributions of D5 and D2 receptor subtypes were not significantly altered by cocaine treatment. These data imply that D5 receptors are predominantly cytoplasmic, D2 receptors are diffusely distributed within the cell, whereas D1 receptors are mostly localized to lipid rafts within the rat frontal cortex. Dopamine receptor subtype localization is susceptible to modulation by pharmacological manipulations that elevate synaptic dopamine, however the functional implications of such drug-induced receptor warrant further investigation. PMID:21236347

  14. Angiotensin type 1 (AT1) and type 2 (AT2) receptors mediate the increase in TGF-beta1 in thyroid hormone-induced cardiac hypertrophy.

    PubMed

    Diniz, G P; Carneiro-Ramos, M S; Barreto-Chaves, M L M

    2007-04-01

    Increased thyroid hormone (TH) levels are known to induce cardiac hypertrophy. Some studies have provided evidence for a functional link between angiotensin II (ANG II) and transforming growth factor beta1 (TGF-beta1) in the heart, both being able to also induce cardiac hypertrophy. However, the contribution of this growth factor activated directly by TH or indirectly by ANG II in cardiac hypertrophy development remains unknown. To analyze the possible role of TGF-beta1 in cardiac hypertrophy induced by TH and also to evaluate if the TGF-beta1 effect is mediated by ANG II receptors, we employed Wistar rats separated into control, hypothyroid (hypo) and hyperthyroid (T4 - 10) groups combined or not with ANG II receptor blockers (losartan or PD123319). Serum levels of T3 and T4, systolic pressure and heart rate confirmed the thyroid state of the groups. The T4 - 10 group presented a significant increase in cardiac TGF-beta1 levels; however, TGF-beta1 levels in the hypo group did not change in relation to the control. Inhibition of the increase in cardiac TGF-beta1 levels was observed in the groups treated with T4 in association with losartan or PD123319 when compared to the T4 - 10 group. These results demonstrate for the first time the TH-modulated induction of cardiac TGF-beta1 in cardiac hypertrophy, and that this effect is mediated by ANG II receptors. PMID:17206447

  15. Serotonin 5-HT2 Receptors Induce a Long-Lasting Facilitation of Spinal Reflexes Independent of Ionotropic Receptor Activity

    PubMed Central

    Shay, Barbara L.; Sawchuk, Michael; Machacek, David W.; Hochman, Shawn

    2009-01-01

    Dorsal root-evoked stimulation of sensory afferents in the hemisected in vitro rat spinal cord produces reflex output, recorded on the ventral roots. Transient spinal 5-HT2C receptor activation induces a long-lasting facilitation of these reflexes (LLFR) by largely unknown mechanisms. Two Sprague-Dawley substrains were used to characterize network properties involved in this serotonin (5-HT) receptor-mediated reflex plasticity. Serotonin more easily produced LLFR in one substrain and a long-lasting depression of reflexes (LLDR) in the other. Interestingly, LLFR and LLDR were bidirectionally interconvertible using 5-HT2A/2C and 5-HT1A receptor agonists, respectively, regardless of substrain. LLFR was predominantly Aβ afferent fiber mediated, consistent with prominent 5-HT2C receptor expression in the Aβ fiber projection territories (deeper spinal laminae). Reflex facilitation involved an unmasking of polysynaptic pathways and an increased receptive field size. LLFR emerged even when reflexes were evoked three to five times/h, indicating an activity independent induction. Both the NMDA and AMPA/kainate receptor-mediated components of the reflex could be facilitated, and facilitation was dependent on 5-HT receptor activation alone, not on coincident reflex activation in the presence of 5-HT. Selective blockade of GABAA and/or glycine receptors also did not prevent reflex amplification and so are not required for LLFR. Indeed, a more robust response was seen after blockade of spinal inhibition, indicating that inhibitory processes serve to limit reflex amplification. Overall we demonstrate that the serotonergic system has the capacity to induce long-lasting bidirectional changes in reflex strength in a manner that is nonassociative and independent of evoked activity or activation of ionotropic excitatory and inhibitory receptors. PMID:16033939

  16. 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.

  17. A transgenic mouse model of neuroepithelial cell specific inducible overexpression of dopamine D1-receptor.

    PubMed

    Fujimoto, K; Araki, K; McCarthy, D M; Sims, J R; Ren, J Q; Zhang, X; Bhide, P G

    2010-10-27

    Dopamine and its receptors appear in the brain during early embryonic period suggesting a role for dopamine in brain development. In fact, dopamine receptor imbalance resulting from impaired physiological balance between D1- and D2-receptor activities can perturb brain development and lead to persisting changes in brain structure and function. Dopamine receptor imbalance can be produced experimentally using pharmacological or genetic methods. Pharmacological methods tend to activate or antagonize the receptors in all cell types. In the traditional gene knockout models the receptor imbalance occurs during development and also at maturity. Therefore, assaying the effects of dopamine imbalance on specific cell types (e.g. precursor versus postmitotic cells) or at specific periods of brain development (e.g. pre- or postnatal periods) is not feasible in these models. We describe a novel transgenic mouse model based on the tetracycline dependent inducible gene expression system in which dopamine D1-receptor transgene expression is induced selectively in neuroepithelial cells of the embryonic brain at experimenter-chosen intervals of brain development. In this model, doxycycline-induced expression of the transgene causes significant overexpression of the D1-receptor and significant reductions in the incorporation of the S-phase marker bromodeoxyuridine into neuroepithelial cells of the basal and dorsal telencephalon indicating marked effects on telencephalic neurogenesis. The D1-receptor overexpression occurs at higher levels in the medial ganglionic eminence (MGE) than the lateral ganglionic eminence (LGE) or cerebral wall (CW). Moreover, although the transgene is induced selectively in the neuroepithelium, D1-receptor protein overexpression appears to persist in postmitotic cells. The mouse model can be modified for neuroepithelial cell-specific inducible expression of other transgenes or induction of the D1-receptor transgene in other cells in specific brain regions by

  18. Antidiabetic thiazolidinediones inhibit leptin (ob) gene expression in 3T3-L1 adipocytes.

    PubMed Central

    Kallen, C B; Lazar, M A

    1996-01-01

    Lack of leptin (ob) protein causes obesity in mice. The leptin gene product is important for normal regulation of appetite and metabolic rate and is produced exclusively by adipocytes. Leptin mRNA was induced during the adipose conversion of 3T3-L1 cells, which are useful for studying adipocyte differentiation and function under controlled conditions. We studied leptin regulation by antidiabetic thiazolidinedione compounds, which are ligands for the adipocyte-specific nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) that regulates the transcription of other adipocyte-specific genes. Remarkably, leptin gene expression was dramatically repressed within a few hours after thiazolidinedione treatment. The ED50 for inhibition of leptin expression by the thiazolidinedione BRL49653 was between 5 and 50 nM, similar to its Kd for binding to PPARgamma. The relatively weak, nonthiazolidinedione PPAR activator WY 14,643 also inhibited leptin expression, but was approximately 1000 times less potent than BRL49653. These results indicate that antidiabetic thiazolidinediones down-regulate leptin gene expression with potencies that correlate with their abilities to bind and activate PPARgamma. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8650171

  19. Ligand-induced ErbB receptor dimerization

    PubMed Central

    Lemmon, Mark A.

    2009-01-01

    Structural studies have provided important new insights into how ligand binding promotes homodimerization and activation of the EGF receptor and the other members of the ErbB family or receptor tyrosine kinases. These structures have also suggested possible explanations for the unique properties of ErbB2, which has no known ligand and can cause cell transformation (and tumorigenesis) by simple overexpression. In parallel with these advances, studies of the EGF receptor at the cell surface increasingly argue that the structural studies are missing key mechanistic components. This is particularly evident in the structural prediction that EGF binding linked to receptor dimerization should be positively cooperative, whereas cell-surface EGF-binding studies suggest negative cooperativity. In this review, I summarize studies of ErbB receptor extracellular regions in solution and of intact receptors at the cell surface, and attempt to reconcile the differences suggested by the two approaches. By combining results obtained with receptor ‘parts’, it is qualitatively possible to explain some models for the properties of the whole receptor. These considerations underline the need to consider the intact ErbB receptors as intact allosterically regulated enzymes, and to combine cellular and structural studies into a complete picture. PMID:19038249

  20. TRPV1 and PLC Participate in Histamine H4 Receptor-Induced Itch

    PubMed Central

    Jian, Tunyu; Yang, Niuniu; Yang, Yan; Zhu, Chan; Yuan, Xiaolin; Yu, Guang; Wang, Changming; Wang, Zhongli; Shi, Hao; Tang, Min; He, Qian; Lan, Lei; Wu, Guanyi; Tang, Zongxiang

    2016-01-01

    Histamine H4 receptor has been confirmed to play a role in evoking peripheral pruritus. However, the ionic and intracellular signaling mechanism of activation of H4 receptor on the dorsal root ganglion (DRG) neurons is still unknown. By using cell culture and calcium imaging, we studied the underlying mechanism of activation of H4 receptor on the DRG neuron. Immepip dihydrobromide (immepip)—a histamine H4 receptor special agonist under cutaneous injection—obviously induced itch behavior of mice. Immepip-induced scratching behavior could be blocked by TRPV1 antagonist AMG9810 and PLC pathway inhibitor U73122. Application of immepip (8.3–50 μM) could also induce a dose-dependent increase in intracellular Ca2+ ([Ca2+]i) of DRG neurons. We found that 77.8% of the immepip-sensitized DRG neurons respond to the TRPV1 selective agonist capsaicin. U73122 could inhibit immepip-induced Ca2+ responses. In addition, immepip-induced [Ca2+]i increase could be blocked by ruthenium red, capsazepine, and AMG9810; however it could not be blocked by TRPA1 antagonist HC-030031. These results indicate that TRPV1 but not TRPA1 is the important ion channel to induce the DRG neurons' responses in the downstream signaling pathway of histamine H4 receptor and suggest that TRPV1 may be involved in the mechanism of histamine-induced itch response by H4 receptor activation. PMID:26819760

  1. TRPV1 and PLC Participate in Histamine H4 Receptor-Induced Itch.

    PubMed

    Jian, Tunyu; Yang, Niuniu; Yang, Yan; Zhu, Chan; Yuan, Xiaolin; Yu, Guang; Wang, Changming; Wang, Zhongli; Shi, Hao; Tang, Min; He, Qian; Lan, Lei; Wu, Guanyi; Tang, Zongxiang

    2016-01-01

    Histamine H4 receptor has been confirmed to play a role in evoking peripheral pruritus. However, the ionic and intracellular signaling mechanism of activation of H4 receptor on the dorsal root ganglion (DRG) neurons is still unknown. By using cell culture and calcium imaging, we studied the underlying mechanism of activation of H4 receptor on the DRG neuron. Immepip dihydrobromide (immepip)-a histamine H4 receptor special agonist under cutaneous injection-obviously induced itch behavior of mice. Immepip-induced scratching behavior could be blocked by TRPV1 antagonist AMG9810 and PLC pathway inhibitor U73122. Application of immepip (8.3-50 μM) could also induce a dose-dependent increase in intracellular Ca(2+) ([Ca(2+)]i) of DRG neurons. We found that 77.8% of the immepip-sensitized DRG neurons respond to the TRPV1 selective agonist capsaicin. U73122 could inhibit immepip-induced Ca(2+) responses. In addition, immepip-induced [Ca(2+)]i increase could be blocked by ruthenium red, capsazepine, and AMG9810; however it could not be blocked by TRPA1 antagonist HC-030031. These results indicate that TRPV1 but not TRPA1 is the important ion channel to induce the DRG neurons' responses in the downstream signaling pathway of histamine H4 receptor and suggest that TRPV1 may be involved in the mechanism of histamine-induced itch response by H4 receptor activation. PMID:26819760

  2. MODULATION OF ACETAMINOPHEN-INDUCED HEPATOTOXICITY BY THE XENOBIOTIC RECEPTOR CAR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have identified the xenobiotic receptor CAR (constitutive androstane receptor) as a key regulator of acetaminophen metabolism and hepatotoxicity. Known CAR activators as well as high doses of acetaminophen induced expression of three acetaminophen-metabolizing enzymes in wild-type but not in CAR-...

  3. Liver growth factor induces testicular regeneration in EDS-treated rats and increases protein levels of class B scavenger receptors.

    PubMed

    Lobo, M V T; Arenas, M I; Huerta, L; Sacristán, S; Pérez-Crespo, M; Gutiérrez-Adán, A; Díaz-Gil, J J; Lasunción, M A; Martín-Hidalgo, A

    2015-01-15

    The aim of the present work was to determine the effects of liver growth factor (LGF) on the regeneration process of rat testes after chemical castration induced by ethane dimethanesulfonate (EDS) by analyzing some of the most relevant proteins involved in cholesterol metabolism, such as hormone sensitive lipase (HSL), 3β-hydroxysteroid dehydrogenase (3β-HSD), scavenger receptor SR-BI, and other components of the SR family that could contribute to the recovery of steroidogenesis and spermatogenesis in the testis. Sixty male rats were randomized to nontreated (controls) and LGF-treated, EDS-treated, and EDS + LGF-treated groups. Testes were obtained on days 10 (T1), 21 (T2), and 35 (T3) after EDS treatment, embedded in paraffin, and analyzed by immunohistochemistry and Western blot. LGF improved the recovery of the seminiferous epithelia, the appearance of the mature pattern of Leydig cell interstitial distribution, and the expression of mature SR-BI. Moreover, LGF treatment resulted in partial recovery of HSL expression in Leydig cells and spermatogonia. No changes in serum testosterone were observed in control or LGF-treated rats, but in EDS-castrated animals LGF treatment induced a progressive increase in serum testosterone levels and 3β-HSD expression. Based on the pivotal role of SR-BI in the uptake of cholesteryl esters from HDL, it is suggested that the observed effects of LGF would facilitate the provision of cholesterol for sperm cell growth and Leydig cell recovery. PMID:25389365

  4. Neurokinin1 receptors regulate morphine-induced endocytosis and desensitization of mu opioid receptors in CNS neurons

    PubMed Central

    Yu, Y. Joy; Arttamangkul, Seksiri; Evans, Christopher J.; Williams, John T.; von Zastrow, Mark

    2009-01-01

    Mu opioid receptors (MORs) are G protein-coupled receptors (GPCRs) that mediate the physiological effects of endogenous opioid neuropeptides and opiate drugs such as morphine. MORs are co-expressed with neurokinin 1 receptors (NK1Rs) in several regions of the central nervous system (CNS) that control opioid dependence and reward. NK1R activation affects opioid reward specifically, however, and the cellular basis for this specificity is unknown. We found that ligand-induced activation of NK1Rs produces a cell autonomous and non-reciprocal inhibition of MOR endocytosis induced by diverse opioids. Studies using epitope-tagged receptors expressed in cultured striatal neurons and a neuroblastoma cell model indicated that this heterologous regulation is mediated by NK1R-dependent sequestration of arrestins on endosome membranes. First, endocytic inhibition mediated by wild type NK1Rs was overcome in cells over-expressing β-arrestin2, a major arrestin isoform expressed in striatum. Second, NK1R activation promoted sequestration of β-arrestin2 on endosomes, whereas MOR activation did not. Third, heterologous inhibition of MOR endocytosis was prevented by mutational disruption of β-arrestin2 sequestration by NK1Rs. NK1R-mediated regulation of MOR trafficking was associated with reduced opioid-induced desensitization of adenylyl cyclase signaling in striatal neurons. Further, heterologous regulation of MOR trafficking was observed in both amygdala and locus coeruleus neurons that naturally co-express these receptors. These results identify a cell autonomous mechanism that may underlie the highly specific effects of NK1R on opioid signaling and suggest, more generally, that receptor-specific trafficking of arrestins may represent a fundamental mechanism for coordinating distinct GPCR-mediated signals at the level of individual CNS neurons. PMID:19129399

  5. The effects of sigma (σ1) receptor-selective ligands on muscarinic receptor antagonist-induced cognitive deficits in mice

    PubMed Central

    Malik, Maninder; Rangel-Barajas, Claudia; Sumien, Nathalie; Su, Chang; Singh, Meharvan; Chen, Zhenglan; Huang, Ren-Qi; Meunier, Johann; Maurice, Tangui; Mach, Robert H; Luedtke, Robert R

    2015-01-01

    Background and Purpose Cognitive deficits in patients with Alzheimer's disease, Parkinson's disease, traumatic brain injury and stroke often involve alterations in cholinergic signalling. Currently available therapeutic drugs provide only symptomatic relief. Therefore, novel therapeutic strategies are needed to retard and/or arrest the progressive loss of memory. Experimental Approach Scopolamine-induced memory impairment provides a rapid and reversible phenotypic screening paradigm for cognition enhancement drug discovery. Male C57BL/6J mice given scopolamine (1 mg·kg−1) were used to evaluate the ability of LS-1–137, a novel sigma (σ1) receptor-selective agonist, to improve the cognitive deficits associated with muscarinic antagonist administration. Key Results LS-1–137 is a high-affinity (Ki = 3.2 nM) σ1 receptor agonist that is 80-fold selective for σ1, compared with σ2 receptors. LS-1–137 binds with low affinity at D2-like (D2, D3 and D4) dopamine and muscarinic receptors. LS-1–137 was found to partially reverse the learning deficits associated with scopolamine administration using a water maze test and an active avoidance task. LS-1–137 treatment was also found to trigger the release of brain-derived neurotrophic factor from rat astrocytes. Conclusions and Implications The σ1 receptor-selective compound LS-1–137 may represent a novel candidate cognitive enhancer for the treatment of muscarinic receptor-dependent cognitive deficits. PMID:25573298

  6. Serotonin 5-HT(2A) receptor activation induces 2-arachidonoylglycerol release through a phospholipase c-dependent mechanism.

    PubMed

    Parrish, Jason C; Nichols, David E

    2006-11-01

    To date, several studies have demonstrated that phospholipase C-coupled receptors stimulate the production of endocannabinoids, particularly 2-arachidonoylglycerol. There is now evidence that endocannabinoids are involved in phospholipase C-coupled serotonin 5-HT(2A) receptor-mediated behavioral effects in both rats and mice. The main objective of this study was to determine whether activation of the 5-HT(2A) receptor leads to the production and release of the endocannabinoid 2-arachidonoylglycerol. NIH3T3 cells stably expressing the rat 5-HT(2A) receptor were first incubated with [(3)H]-arachidonic acid for 24 h. Following stimulation with 10 mum serotonin, lipids were extracted from the assay medium, separated by thin layer chromatography, and analyzed by liquid scintillation counting. Our results indicate that 5-HT(2A) receptor activation stimulates the formation and release of 2-arachidonoylglycerol. The 5-HT(2A) receptor-dependent release of 2-arachidonoylglycerol was partially dependent on phosphatidylinositol-specific phospholipase C activation. Diacylglycerol produced downstream of 5-HT(2A) receptor-mediated phospholipase D or phosphatidylcholine-specific phospholipase C activation did not appear to contribute to 2-arachidonoylglycerol formation in NIH3T3-5HT(2A) cells. In conclusion, our results support a functional model where neuromodulatory neurotransmitters such as serotonin may act as regulators of endocannabinoid tone at excitatory synapses through the activation of phospholipase C-coupled G-protein coupled receptors. PMID:17010161

  7. Roles of parathyroid hormone (PTH) receptor and reactive oxygen species in hyperlipidemia-induced PTH resistance in preosteoblasts.

    PubMed

    Li, Xin; Garcia, Jamie; Lu, Jinxiu; Iriana, Sidney; Kalajzic, Ivo; Rowe, David; Demer, Linda L; Tintut, Yin

    2014-01-01

    Bioactive lipids initiate inflammatory reactions leading to pathogenesis of atherosclerosis. Evidence shows that they also contribute to bone loss by inhibiting parathyroid hormone receptor (PTH1R) expression and differentiation of osteoblasts. We previously demonstrated that bone anabolic effects of PTH(1-34) are blunted in hyperlipidemic mice and that these PTH effects are restored by antioxidants. However, it is not clear which osteoblastic cell developmental stage is targeted by bioactive lipids. To investigate the effects of hyperlipidemia at the cellular level, hyperlipidemic Ldlr(-/-) mice were bred with Col3.6GFPtpz mice, in which preosteoblasts/osteoblasts carry a topaz fluorescent label, and with Col2.3GFPcyan mice, in which more mature osteoblasts/osteocytes carry a cyan fluorescent label. Histological analyses of trabecular bone surfaces in femoral as well as calvarial bones showed that intermittent PTH(1-34) increased fluorescence intensity in WT-Tpz mice, but not in Tpz-Ldlr(-/-) mice. In contrast, PTH(1-34) did not alter fluorescence intensity in femoral cortical envelopes of either WT-Cyan or Ldlr(-/-)-Cyan mice. To test the mechanism of PTH1R downregulation, preosteoblastic MC3T3-E1 cells were treated with bioactive lipids and the antioxidant Trolox. Results showed that inhibitory effects of PTH1R levels by bioactive lipids were rescued by pretreatment with Trolox. The inhibitory effects on expression of PTH1R as well as on PTH-induced osteoblastic genes were mimicked by xanthine/xanthine oxidase, a known generator of reactive oxygen species. These findings suggest an important role of the preosteoblastic development stage as the target and downregulation of PTH receptor expression mediated by intracellular oxidant stress as a mechanism in hyperlipidemia-induced PTH resistance. PMID:24038594

  8. GRK2 Protein-mediated Transphosphorylation Contributes to Loss of Function of μ-Opioid Receptors Induced by Neuropeptide FF (NPFF2) Receptors*

    PubMed Central

    Moulédous, Lionel; Froment, Carine; Dauvillier, Stéphanie; Burlet-Schiltz, Odile; Zajac, Jean-Marie; Mollereau, Catherine

    2012-01-01

    Neuropeptide FF (NPFF) interacts with specific receptors to modulate opioid functions in the central nervous system. On dissociated neurons and neuroblastoma cells (SH-SY5Y) transfected with NPFF receptors, NPFF acts as a functional antagonist of μ-opioid (MOP) receptors by attenuating the opioid-induced inhibition of calcium conductance. In the SH-SY5Y model, MOP and NPFF2 receptors have been shown to heteromerize. To understand the molecular mechanism involved in the anti-opioid activity of NPFF, we have investigated the phosphorylation status of the MOP receptor using phospho-specific antibody and mass spectrometry. Similarly to direct opioid receptor stimulation, activation of the NPFF2 receptor by [d-Tyr-1-(NMe)Phe-3]NPFF (1DMe), an analog of NPFF, induced the phosphorylation of Ser-377 of the human MOP receptor. This heterologous phosphorylation was unaffected by inhibition of second messenger-dependent kinases and, contrarily to homologous phosphorylation, was prevented by inactivation of Gi/o proteins by pertussis toxin. Using siRNA knockdown we could demonstrate that 1DMe-induced Ser-377 cross-phosphorylation and MOP receptor loss of function were mediated by the G protein receptor kinase GRK2. In addition, mass spectrometric analysis revealed that the phosphorylation pattern of MOP receptors was qualitatively similar after treatment with the MOP agonist Tyr-d-Ala-Gly (NMe)-Phe-Gly-ol (DAMGO) or after treatment with the NPFF agonist 1DMe, but the level of multiple phosphorylation was more intense after DAMGO. Finally, NPFF2 receptor activation was sufficient to recruit β-arrestin2 to the MOP receptor but not to induce its internalization. These data show that NPFF-induced heterologous desensitization of MOP receptor signaling is mediated by GRK2 and could involve transphosphorylation within the heteromeric receptor complex. PMID:22375000

  9. GRK2 protein-mediated transphosphorylation contributes to loss of function of μ-opioid receptors induced by neuropeptide FF (NPFF2) receptors.

    PubMed

    Moulédous, Lionel; Froment, Carine; Dauvillier, Stéphanie; Burlet-Schiltz, Odile; Zajac, Jean-Marie; Mollereau, Catherine

    2012-04-13

    Neuropeptide FF (NPFF) interacts with specific receptors to modulate opioid functions in the central nervous system. On dissociated neurons and neuroblastoma cells (SH-SY5Y) transfected with NPFF receptors, NPFF acts as a functional antagonist of μ-opioid (MOP) receptors by attenuating the opioid-induced inhibition of calcium conductance. In the SH-SY5Y model, MOP and NPFF(2) receptors have been shown to heteromerize. To understand the molecular mechanism involved in the anti-opioid activity of NPFF, we have investigated the phosphorylation status of the MOP receptor using phospho-specific antibody and mass spectrometry. Similarly to direct opioid receptor stimulation, activation of the NPFF(2) receptor by [D-Tyr-1-(NMe)Phe-3]NPFF (1DMe), an analog of NPFF, induced the phosphorylation of Ser-377 of the human MOP receptor. This heterologous phosphorylation was unaffected by inhibition of second messenger-dependent kinases and, contrarily to homologous phosphorylation, was prevented by inactivation of G(i/o) proteins by pertussis toxin. Using siRNA knockdown we could demonstrate that 1DMe-induced Ser-377 cross-phosphorylation and MOP receptor loss of function were mediated by the G protein receptor kinase GRK2. In addition, mass spectrometric analysis revealed that the phosphorylation pattern of MOP receptors was qualitatively similar after treatment with the MOP agonist Tyr-D-Ala-Gly (NMe)-Phe-Gly-ol (DAMGO) or after treatment with the NPFF agonist 1DMe, but the level of multiple phosphorylation was more intense after DAMGO. Finally, NPFF(2) receptor activation was sufficient to recruit β-arrestin2 to the MOP receptor but not to induce its internalization. These data show that NPFF-induced heterologous desensitization of MOP receptor signaling is mediated by GRK2 and could involve transphosphorylation within the heteromeric receptor complex. PMID:22375000

  10. Desensitization of GABAergic receptors as a mechanism of zolpidem-induced somnambulism.

    PubMed

    Juszczak, Grzegorz R

    2011-08-01

    Sleepwalking is a frequently reported side effect of zolpidem which is a short-acting hypnotic drug potentiating activity of GABA(A) receptors. Paradoxically, the most commonly used medications for somnambulism are benzodiazepines, especially clonazepam, which also potentiate activity of GABA(A) receptors. It is proposed that zolpidem-induced sleepwalking can be explained by the desensitization of GABAergic receptors located on serotonergic neurons. According to the proposed model, the delay between desensitization of GABA receptors and a compensatory decrease in serotonin release constitutes the time window for parasomnias. The occurrence of sleepwalking depends on individual differences in receptor desensitization, autoregulation of serotonin release and drug pharmacokinetics. The proposed mechanism of interaction between GABAergic and serotonergic systems can be also relevant for zolpidem abuse and zolpidem-induced hallucinations. It is therefore suggested that special care should be taken when zolpidem is used in patients taking at the same time selective serotonin reuptake inhibitors. PMID:21565448