Science.gov

Sample records for hex regulates hepatocyte

  1. Control of Proteobacterial Central Carbon Metabolism by the HexR Transcriptional Regulator. A Case Study in Shewanella oneidensis

    SciTech Connect

    Leyn, Semen; Li, Xiaoqing; Zheng, Qijing; Novichkov, Pavel; Reed, Samantha B.; Romine, Margaret F.; Fredrickson, Jim K.; Yang, Chen; Osterman, Andrei L.; Rodionov, Dmitry A.

    2011-08-17

    Bacteria exploit multiple mechanisms for controlling central carbon metabolism (CCM). Thus, a bioinformatic analysis together with some experimental data implicated HexR transcriptional factor as a global CCM regulator in some lineages of Gammaproteobacteria operating as a functional replacement of Cra regulator characteristic of Enterobacteriales. In this study we combined a large-scale comparative genomic reconstruction of HexRcontrolled regulons in 87 species of Proteobacteria with the detailed experimental analysis of HexR regulatory network in Shewanella oneidensis model system. Although nearly all of the HexR-controlled genes are associated with CCM, remarkable variations were revealed in the scale (from 1-2 target operons in Enterobacteriales up to 20 operons in Aeromonadales) and gene content of HexR regulons between 11 compared lineages. A predicted 17-bp pseudo-palindrome with a consensus tTGTAATwwwATTACa, was confirmed as HexR-binding motif for 15 target operons (comprising 30 genes) by in vitro binding assays. The negative effect of the key CCM intermediate, 2-keto-3-deoxy-6- phosphogluconate, on the DNA-regulator complex formation was verified. A dual mode of HexR action on various target promoters, repression of genes involved in catabolic pathways and activation of gluconeogenic genes, was for the first time predicted by the bioinformatc analysis and experimentally verified by changed gene expression pattern in S. oneidensis AhexR mutant. Phenotypic profiling revealed the inability of this mutant to grow on lactate or pyruvate as a single carbon source. A comparative metabolic flux analysis of wild-type and mutant strains of S. oneidensis using 13Clactate labeling and GC-MS analysis confirmed the hypothesized HexR role as a master regulator of gluconeogenic flux from pyruvate via the transcriptional activation of phosphoenolpyruvate synthase (PpsA).

  2. Hormonal regulation of hepatocyte tight junctional permeability

    SciTech Connect

    Lowe, P.J.; Miyai, K.; Steinbach, J.H.; Hardison, W.G.M. Univ. of California, San Diego )

    1988-10-01

    The authors have investigated the effects of hormones on the permeability of the hepatocyte tight junction to two probes, ({sup 14}C)sucrose and horseradish peroxidase, using one-pass perfused rat livers. Using a single injection of horseradish peroxidase the authors have demonstrated that this probe can enter bile by two pathways that are kinetically distinct, a fast pathway, which corresponds to the passage of the probe through the hepatocyte tight junctions, and a slow pathway, which corresponds to the transcytotic entry into bile. The passage of horseradish peroxidase through the hepatocyte tight junctions was confirmed by electron microscopic histochemistry. Vasopressin, epinephrine, and angiotensin II, hormones that act in the hepatocyte through the intracellular mediators calcium, the inositol polyphosphates, and diacylglycerol, increased the bile-to-perfusion fluid ratio of ({sup 14}C)sucrose and the rapid entry of horseradish peroxidase into bile, indicating that the permeability of the tight junctions to these probes was increased. The effect of these hormones was dose dependent and in the cases of angiotensin II and epinephrine was inhibited by the specific inhibitors (Sar{sup 1},Thr{sup 8})angiotensin II and prazosin, respectively. Dibutyryl adenosine 3{prime},5{prime}-cyclic monophosphate did not affect the ({sup 14}C)sucrose bile-to-perfusion fluid ratio or the fast entry of horseradish peroxidase into bile. These results suggest that the hepatocyte tight junction can no longer be considered a static system of pores separating blood from bile. It is rather a dynamic barrier potentially capable of influencing the composition of the bile.

  3. Chemokine Receptors, CXCR1 and CXCR2, Differentially Regulate Exosome Release in Hepatocytes

    PubMed Central

    Nojima, Hiroyuki; Konishi, Takanori; Freeman, Christopher M.; Schuster, Rebecca M.; Japtok, Lukasz; Kleuser, Burkhard; Edwards, Michael J.; Gulbins, Erich; Lentsch, Alex B.

    2016-01-01

    Exosomes are small membrane vesicles released by different cell types, including hepatocytes, that play important roles in intercellular communication. We have previously demonstrated that hepatocyte-derived exosomes contain the synthetic machinery to form sphingosine-1-phosphate (S1P) in target hepatocytes resulting in proliferation and liver regeneration after ischemia/reperfusion (I/R) injury. We also demonstrated that the chemokine receptors, CXCR1 and CXCR2, regulate liver recovery and regeneration after I/R injury. In the current study, we sought to determine if the regulatory effects of CXCR1 and CXCR2 on liver recovery and regeneration might occur via altered release of hepatocyte exosomes. We found that hepatocyte release of exosomes was dependent upon CXCR1 and CXCR2. CXCR1-deficient hepatocytes produced fewer exosomes, whereas CXCR2-deficient hepatocytes produced more exosomes compared to their wild-type controls. In CXCR2-deficient hepatocytes, there was increased activity of neutral sphingomyelinase (Nsm) and intracellular ceramide. CXCR1-deficient hepatocytes had no alterations in Nsm activity or ceramide production. Interestingly, exosomes from CXCR1-deficient hepatocytes had no effect on hepatocyte proliferation, due to a lack of neutral ceramidase and sphingosine kinase. The data demonstrate that CXCR1 and CXCR2 regulate hepatocyte exosome release. The mechanism utilized by CXCR1 remains elusive, but CXCR2 appears to modulate Nsm activity and resultant production of ceramide to control exosome release. CXCR1 is required for packaging of enzymes into exosomes that mediate their hepatocyte proliferative effect. PMID:27551720

  4. Chemokine Receptors, CXCR1 and CXCR2, Differentially Regulate Exosome Release in Hepatocytes.

    PubMed

    Nojima, Hiroyuki; Konishi, Takanori; Freeman, Christopher M; Schuster, Rebecca M; Japtok, Lukasz; Kleuser, Burkhard; Edwards, Michael J; Gulbins, Erich; Lentsch, Alex B

    2016-01-01

    Exosomes are small membrane vesicles released by different cell types, including hepatocytes, that play important roles in intercellular communication. We have previously demonstrated that hepatocyte-derived exosomes contain the synthetic machinery to form sphingosine-1-phosphate (S1P) in target hepatocytes resulting in proliferation and liver regeneration after ischemia/reperfusion (I/R) injury. We also demonstrated that the chemokine receptors, CXCR1 and CXCR2, regulate liver recovery and regeneration after I/R injury. In the current study, we sought to determine if the regulatory effects of CXCR1 and CXCR2 on liver recovery and regeneration might occur via altered release of hepatocyte exosomes. We found that hepatocyte release of exosomes was dependent upon CXCR1 and CXCR2. CXCR1-deficient hepatocytes produced fewer exosomes, whereas CXCR2-deficient hepatocytes produced more exosomes compared to their wild-type controls. In CXCR2-deficient hepatocytes, there was increased activity of neutral sphingomyelinase (Nsm) and intracellular ceramide. CXCR1-deficient hepatocytes had no alterations in Nsm activity or ceramide production. Interestingly, exosomes from CXCR1-deficient hepatocytes had no effect on hepatocyte proliferation, due to a lack of neutral ceramidase and sphingosine kinase. The data demonstrate that CXCR1 and CXCR2 regulate hepatocyte exosome release. The mechanism utilized by CXCR1 remains elusive, but CXCR2 appears to modulate Nsm activity and resultant production of ceramide to control exosome release. CXCR1 is required for packaging of enzymes into exosomes that mediate their hepatocyte proliferative effect. PMID:27551720

  5. Regulation of Hepatocyte Fate by Interferon-γ

    PubMed Central

    Horras, Christopher J.; Lamb, Cheri L.; Mitchell, Kristen A.

    2011-01-01

    Interferon (IFN)-γ is a cytokine known for its immunomodulatory and anti-proliferative action. In the liver, IFN-γ can induce hepatocyte apoptosis or inhibit hepatocyte cell cycle progression. This article reviews recent mechanistic reports that describe how IFN-γ may direct the fate of hepatocytes either towards apoptosis or a cell cycle arrest. This review also describes a probable role for IFN-γ in modulating hepatocyte fate during liver regeneration, transplantation, hepatitis, fibrosis and hepatocellular carcinoma, and highlights promising areas of research that may lead to the development of IFN-γ as a therapy to enhance recovery from liver disease. PMID:21334249

  6. WNT-3A Regulates an Axin1/NRF2 Complex That Regulates Antioxidant Metabolism in Hepatocytes

    PubMed Central

    Rada, Patricia; Rojo, Ana I.; Offergeld, Anika; Feng, Gui Jie; Velasco-Martín, Juan P.; González-Sancho, José Manuel; Valverde, Ángela M.; Dale, Trevor; Regadera, Javier

    2015-01-01

    Abstract Aims: Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a master regulator of oxidant and xenobiotic metabolism, but it is unknown how it is regulated to provide basal expression of this defense system. Here, we studied the putative connection between NRF2 and the canonical WNT pathway, which modulates hepatocyte metabolism. Results: WNT-3A increased the levels of NRF2 and its transcriptional signature in mouse hepatocytes and HEK293T cells. The use of short interfering RNAs in hepatocytes and mouse embryonic fibroblasts which are deficient in the redox sensor Kelch-like ECH-associated protein 1 (KEAP1) indicated that WNT-3A activates NRF2 in a β-Catenin- and KEAP1-independent manner. WNT-3A stabilized NRF2 by preventing its GSK-3-dependent phosphorylation and subsequent SCF/β-TrCP-dependent ubiquitination and proteasomal degradation. Axin1 and NRF2 were physically associated in a protein complex that was regulated by WNT-3A, involving the central region of Axin1 and the Neh4/Neh5 domains of NRF2. Axin1 knockdown increased NRF2 protein levels, while Axin1 stabilization with Tankyrase inhibitors blocked WNT/NRF2 signaling. The relevance of this novel pathway was assessed in mice with a conditional deletion of Axin1 in the liver, which showed upregulation of the NRF2 signature in hepatocytes and disruption of liver zonation of antioxidant metabolism. Innovation: NRF2 takes part in a protein complex with Axin1 that is regulated by the canonical WNT pathway. This new WNT-NRF2 axis controls the antioxidant metabolism of hepatocytes. Conclusion: These results uncover the participation of NRF2 in a WNT-regulated signalosome that participates in basal maintenance of hepatic antioxidant metabolism. Antioxid. Redox Signal. 22, 555–571. PMID:25336178

  7. The Game of Hex

    ERIC Educational Resources Information Center

    Scott, Paul

    2007-01-01

    In this article, the author discusses the game of Hex, including its history, strategies and problems. Like all good games, the rules are very simple. Hex is played on a diamond shaped board made up of hexagons. It can be of any size, but an 11x11 board makes for a good game. Two opposite sides of the diamond are labelled "red," the other two…

  8. The LysR Transcription Factor, HexS, Is Required for Glucose Inhibition of Prodigiosin Production by Serratia marcescens.

    PubMed

    Stella, Nicholas A; Fender, James E; Lahr, Roni M; Kalivoda, Eric J; Shanks, Robert M Q

    2012-12-01

    Generation of many useful microbe-derived secondary metabolites, including the red pigment prodigiosin of the bacterium Serratia marcescens, is inhibited by glucose. In a previous report, a genetic approach was used to determine that glucose dehydrogenase activity (GDH) is required for inhibiting prodigiosin production and transcription of the prodigiosin biosynthetic operon (pigA-N). However, the transcription factor(s) that regulate this process were not characterized. Here we tested the hypothesis that HexS, a LysR-family transcription factor similar to LrhA of Escherichia coli, is required for inhibition of prodigiosin by growth in glucose. We observed that mutation of the hexS gene in S. marcescens allowed the precocious production of prodigiosin in glucose-rich medium conditions that completely inhibited prodigiosin production by the wild type. Unlike previously described mutants able to generate prodigiosin in glucose-rich medium, hexS mutants exhibited GDH activity and medium acidification similar to the wild type. Glucose inhibittion of pigA expression was shown to be dependent upon HexS, suggesting that HexS is a key transcription factor in secondary metabolite regulation in response to medium pH. These data give insight into the prodigiosin regulatory pathway and could be used to enhance the production of secondary metabolites. PMID:24358451

  9. Hex ball torque test

    NASA Technical Reports Server (NTRS)

    Robinson, B. A.; Foster, C. L.

    1986-01-01

    A series of torque tests were performed on four flight-type hex ball universal joints in order to characterize and determine the actual load-carrying capability of this device. The universal joint is a part of manual actuation rods for scientific instruments within the Hubble Space Telescope. It was found that the hex ball will bind slightly during the initial load application. This binding did not affect the function of the universal joint, and the units would wear-in after a few additional loading cycles. The torsional yield load was approximately 50 ft-lb, and was consistent among the four test specimens. Also, the torque required to cause complete failure exceeded 80 ft-lb. It is concluded that the hex ball universal joint is suitable for its intended applications.

  10. Hormonal regulation of mannan-binding lectin synthesis in hepatocytes

    PubMed Central

    Sørensen, C M; Hansen, T K; Steffensen, R; Jensenius, J C; Thiel, S

    2006-01-01

    Activation of the complement system via the plasma protein mannan-binding lectin (MBL) provides a first line of defence against infections. The plasma level of MBL is, in part, determined genetically, but may also be influenced by different hormones in vivo. Here we study the hormonal regulation of MBL synthesis from the human hepatocyte cell line HuH-7. Cells were exposed to medium with growth hormone (GH), hydrocortisone, insulin-like growth factor (IGF)-1, insulin, interleukin (IL)-6 or thyroid hormones (T3 or T4). After 3 days the concentration of MBL in the culture supernatants was determined and the amount of mRNA for MBL was measured, relative to mRNA for β2 microglobulin. GH, IL-6, T3 and T4 significantly increased MBL synthesis in a dose-dependent manner, while hydrocortisone, insulin and IGF-1 had no effect. T3 caused a fourfold increase at 1 nM of T3 (P < 0·001) and at 100 nM of T3 the production was increased more than eightfold. The effect of T4 was less potent, reaching an eightfold increase at 1 µM of T4 (P < 0·001). GH augmented the production of MBL threefold at a concentration of 100 ng/ml (P = 0·018) with no further effect up to 10 µg/ml, whereas IL-6 caused only a very weak increase in MBL production. MBL mRNA levels were stable during the first 24 h of T3 stimulation but increased significantly between 24 and 48 h. The results suggest that MBL synthesis in humans may be increased by thyroid hormone and GH, whereas it does not exhibit a classical IL-6-dependent response. PMID:16792688

  11. SRN1, a yeast gene involved in RNA processing, is identical to HEX2/REG1, a negative regulator in glucose repression.

    PubMed Central

    Tung, K S; Norbeck, L L; Nolan, S L; Atkinson, N S; Hopper, A K

    1992-01-01

    The yeast RNA1 gene encodes a cytosolic protein that affects pre-tRNA splicing, pre-rRNA processing, the production of mRNA, and the export of RNA from the nucleus to the cytosol. In an attempt to understand how the RNA1 protein affects such a diverse set of processes, we sought second-site suppressors of a mutation, rna1-1, of the RNA1 locus. Mutations in a single complementation group were obtained. These lesions proved to be in the same gene, SRN1, identified previously in a search for second-site suppressors of mutations that affect the removal of intervening sequences from pre-mRNAs. The SRN1 gene was mapped, cloned, and sequenced. DNA sequence analysis and the phenotype of disruption mutations showed that, surprisingly, SRN1 is identical to HEX2/REG1, a gene that negatively regulates glucose-repressible genes. Interestingly, SRN1 is not a negative regulator of RNA1 at the transcriptional, translational, or protein stability level. However, SRN1 does regulate the level of two newly discovered antigens, p43 and p70, one of which is not glucose repressible. These studies for the first time link RNA processing and carbon catabolite repression. Images PMID:1588964

  12. Hormonal regulation of fibrinogen synthesis in cultured hepatocytes.

    PubMed

    Grieninger, G; Plant, P W; Liang, T J; Kalb, R G; Amrani, D; Mosesson, M W; Hertzberg, K M; Pindyck, J

    1983-06-27

    Most of what was originally known of the effects of hormones on fibrinogen synthesis was based, as noted above, on experiments involving surgical removal of endocrine glands. Some caution should be exercised when using such in vivo experiments to derive the hormonal requirements of fibrinogen synthesis, however, since multiple hormonal alterations often occur in these animals. The development of a variety of ex vivo systems has allowed investigators to more carefully control the hepatocellular environment. The work of several laboratories, including our own, has now made it clear that hormones and other agents directly stimulate hepatocellular synthesis of fibrinogen. From the studies summarized here, using chick embryo hepatocytes as a model, several generalizations emerge: Fibrinogen synthesis may be considered to be a "constitutive" liver function, since hepatocytes cultured without serum, hormones or other macromolecular supplements synthesize this protein at a basal rate for several days. Addition of certain hormones (e.g. T3, dexamethasone, insulin), individually and in physiological concentrations, elicits an increase in fibrinogen production, varying with each agent in onset, dose, minimum exposure required and accompanying effects on the synthesis of other plasma proteins. Glucocorticoids and thyroid hormones are similar in the selectivity of their stimulation (neither affects albumin or transferrin synthesis) but differ in that thyroid hormones need to be present for just a short "triggering" period. The stimulation of fibrinogen synthesis by insulin occurs only following prolonged exposure to concentrations 10-times higher than the very low doses to which albumin synthesis responds rapidly. PMID:6307104

  13. 5α-Reductase Type 2 Regulates Glucocorticoid Action and Metabolic Phenotype in Human Hepatocytes.

    PubMed

    Nasiri, Maryam; Nikolaou, Nikolaos; Parajes, Silvia; Krone, Nils P; Valsamakis, George; Mastorakos, George; Hughes, Beverly; Taylor, Angela; Bujalska, Iwona J; Gathercole, Laura L; Tomlinson, Jeremy W

    2015-08-01

    Glucocorticoids and androgens have both been implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD); androgen deficiency in males, androgen excess in females, and glucocorticoid excess in both sexes are associated with NAFLD. Glucocorticoid and androgen action are regulated at a prereceptor level by the enzyme 5α-reductase type 2 (SRD5A2), which inactivates glucocorticoids to their dihydrometabolites and converts T to DHT. We have therefore explored the role of androgens and glucocorticoids and their metabolism by SRD5A2 upon lipid homeostasis in human hepatocytes. In both primary human hepatocytes and human hepatoma cell lines, glucocorticoids decreased de novo lipogenesis in a dose-dependent manner. Whereas androgen treatment (T and DHT) increased lipogenesis in cell lines and in primary cultures of human hepatocytes from female donors, it was without effect in primary hepatocyte cultures from men. SRD5A2 overexpression reduced the effects of cortisol to suppress lipogenesis and this effect was lost following transfection with an inactive mutant construct. Conversely, pharmacological inhibition using the 5α-reductase inhibitors finasteride and dutasteride augmented cortisol action. We have demonstrated that manipulation of SRD5A2 activity can regulate lipogenesis in human hepatocytes in vitro. This may have significant clinical implications for those patients prescribed 5α-reductase inhibitors, in particular augmenting the actions of glucocorticoids to modulate hepatic lipid flux. PMID:25974403

  14. 5α-Reductase Type 2 Regulates Glucocorticoid Action and Metabolic Phenotype in Human Hepatocytes

    PubMed Central

    Nasiri, Maryam; Nikolaou, Nikolaos; Parajes, Silvia; Krone, Nils P.; Valsamakis, George; Mastorakos, George; Hughes, Beverly; Taylor, Angela; Bujalska, Iwona J.; Gathercole, Laura L.

    2015-01-01

    Glucocorticoids and androgens have both been implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD); androgen deficiency in males, androgen excess in females, and glucocorticoid excess in both sexes are associated with NAFLD. Glucocorticoid and androgen action are regulated at a prereceptor level by the enzyme 5α-reductase type 2 (SRD5A2), which inactivates glucocorticoids to their dihydrometabolites and converts T to DHT. We have therefore explored the role of androgens and glucocorticoids and their metabolism by SRD5A2 upon lipid homeostasis in human hepatocytes. In both primary human hepatocytes and human hepatoma cell lines, glucocorticoids decreased de novo lipogenesis in a dose-dependent manner. Whereas androgen treatment (T and DHT) increased lipogenesis in cell lines and in primary cultures of human hepatocytes from female donors, it was without effect in primary hepatocyte cultures from men. SRD5A2 overexpression reduced the effects of cortisol to suppress lipogenesis and this effect was lost following transfection with an inactive mutant construct. Conversely, pharmacological inhibition using the 5α-reductase inhibitors finasteride and dutasteride augmented cortisol action. We have demonstrated that manipulation of SRD5A2 activity can regulate lipogenesis in human hepatocytes in vitro. This may have significant clinical implications for those patients prescribed 5α-reductase inhibitors, in particular augmenting the actions of glucocorticoids to modulate hepatic lipid flux. PMID:25974403

  15. Role of apoptotic hepatocytes in HCV dissemination: regulation by acetaldehyde.

    PubMed

    Ganesan, Murali; Natarajan, Sathish Kumar; Zhang, Jinjin; Mott, Justin L; Poluektova, Larisa I; McVicker, Benita L; Kharbanda, Kusum K; Tuma, Dean J; Osna, Natalia A

    2016-06-01

    Alcohol consumption exacerbates hepatitis C virus (HCV) pathogenesis and promotes disease progression, although the mechanisms are not quite clear. We have previously observed that acetaldehyde (Ach) continuously produced by the acetaldehyde-generating system (AGS), temporarily enhanced HCV RNA levels, followed by a decrease to normal or lower levels, which corresponded to apoptosis induction. Here, we studied whether Ach-induced apoptosis caused depletion of HCV-infected cells and what role apoptotic bodies (AB) play in HCV-alcohol crosstalk. In liver cells exposed to AGS, we observed the induction of miR-122 and miR-34a. As miR-34a has been associated with apoptotic signaling and miR-122 with HCV replication, these findings may suggest that cells with intensive viral replication undergo apoptosis. Furthermore, when AGS-induced apoptosis was blocked by a pan-caspase inhibitor, the expression of HCV RNA was not changed. AB from HCV-infected cells contained HCV core protein and the assembled HCV particle that infect intact hepatocytes, thereby promoting the spread of infection. In addition, AB are captured by macrophages to switch their cytokine profile to the proinflammatory one. Macrophages exposed to HCV(+) AB expressed more IL-1β, IL-18, IL-6, and IL-10 mRNAs compared with those exposed to HCV(-) AB. The generation of AB from AGS-treated HCV-infected cells even enhanced the induction of aforementioned cytokines. We conclude that HCV and alcohol metabolites trigger the formation of AB containing HCV particles. The consequent spread of HCV to neighboring hepatocytes via infected AB, as well as the induction of liver inflammation by AB-mediated macrophage activation potentially exacerbate the HCV infection course by alcohol and worsen disease progression. PMID:27056722

  16. Involvement of Hex in the initiation of feather morphogenesis.

    PubMed

    Obinata, Akiko; Akimoto, Yoshihiro

    2005-01-01

    In a previous study, we showed that the proline-rich divergent homeobox gene Hex/Prh is expressed in dorsal skin of the chick embryo before and during feather bud development and that the pattern of Hex mRNA expression in the epidermis is similar to that of Wnt7a mRNA. In order to study the function of Hex and the relationship between Hex and Wnt7a in feather bud development, sense and/or antisense sequences of Hex or Wnt7a were ectopically and transiently expressed in the dorsal skin with the epidermal side toward the cathode by electroporation at the placode stage and then the skin was cultured. Increased expression of Wnt7a and beta-catenin mRNA was observed in the same region where Hex-EGFP fusion protein was expressed 2 days after culture, which was followed by extra bud formation a few days later as a result of the stimulation of cell proliferation. Concomitantly, expression of Notch1 mRNA, which is expressed in normal bud development, increased in Hex-overexpressing skin. However, ectopic Wnt7a expression induced neither Hex expression nor extra bud formation in normal skin. Antisense Wnt7a specifically inhibited bud initiation in Hex-overexpressing skin but did not in normal skin. Taken together, these results suggest that Hex is upstream of Wnt7a and beta-catenin and regulates the Wnt signaling pathway in feather bud initiation and that some other Wnt signals in addition to Wnt7a may be required for bud initiation. PMID:16281172

  17. Regulation of host innate immunity by hepatitis C virus: crosstalk between hepatocyte and NK/DC

    PubMed Central

    Park, Sung-Jae; Hahn, Young S.

    2014-01-01

    Hepatitis C virus (HCV) infection in humans is remarkably efficient in establishing viral persistence, leading to the development of liver cirrhosis and hepatocellular carcinoma. CD8+ T cells are involved in controlling HCV infection; but, in chronic HCV patients, severe CD4+ and CD8+ T cell dysfunction has been observed. This suggests that HCV may employ numerous mechanisms to counteract or possibly suppress the host T cell responses. The primary site of HCV replication occurs within hepatocytes in the liver. As a result of liver enodothelial cells perforated by fenestrations, parenchymal cells (hepatocytes) are not separated by a basal membrane, and thereby HCV-infected hepatocytes are extensively capable of interacting with innate immune cells including NK, DC. Recent studies reveal that the function of NK and DC function is significantly impaired in chronic HCV patients. Given a critical role of NK and DC in limiting HCV replication at the early phase of viral infection, it is likely that HCV-infected hepatocytes might be responsible for impairing NK and DC function by enhancing the expression of immunoregulatory molecules (either soluble or cell surface). Thus, this impairment of innate immunity attributes to the failure of generating effective T cell responses to clear HCV infection. In this article, we will review studies highlighting the regulation of innate immunity by HCV and crosstalk between hepatocytes and NK/DC in the hepatic environment. PMID:24688607

  18. The Machine Intelligence Hex Project

    ERIC Educational Resources Information Center

    Chalup, Stephan K.; Mellor, Drew; Rosamond, Fran

    2005-01-01

    Hex is a challenging strategy board game for two players. To enhance students' progress in acquiring understanding and practical experience with complex machine intelligence and programming concepts we developed the Machine Intelligence Hex (MIHex) project. The associated undergraduate student assignment is about designing and implementing Hex…

  19. Hepatocyte β-Klotho regulates lipid homeostasis but not body weight in mice.

    PubMed

    Kobayashi, Kanako; Tanaka, Tomohiro; Okada, Sadanori; Morimoto, Yuki; Matsumura, Shigenobu; Manio, Mark Christian C; Inoue, Kazuo; Kimura, Kumi; Yagi, Takashi; Saito, Yoshihiko; Fushiki, Tohru; Inoue, Hiroshi; Matsumoto, Michihiro; Nabeshima, Yo-Ichi

    2016-02-01

    β-Klotho (β-Kl), a transmembrane protein expressed in the liver, pancreas, adipose tissues, and brain, is essential for feedback suppression of hepatic bile acid synthesis. Because bile acid is a key regulator of lipid and energy metabolism, we hypothesized potential and tissue-specific roles of β-Kl in regulating plasma lipid levels and body weight. By crossing β-kl(-/-) mice with newly developed hepatocyte-specific β-kl transgenic (Tg) mice, we generated mice expressing β-kl solely in hepatocytes (β-kl(-/-)/Tg). Gene expression, metabolomic, and in vivo flux analyses consistently revealed that plasma level of cholesterol, which is over-excreted into feces as bile acids in β-kl(-/-), is maintained in β-kl(-/-) mice by enhanced de novo cholesterogenesis. No compensatory increase in lipogenesis was observed, despite markedly decreased plasma triglyceride. Along with enhanced bile acid synthesis, these lipid dysregulations in β-kl(-/-) were completely reversed in β-kl(-/-)/Tg mice. In contrast, reduced body weight and resistance to diet-induced obesity in β-kl(-/-) mice were not reversed by hepatocyte-specific restoration of β-Kl expression. We conclude that β-Kl in hepatocytes is necessary and sufficient for lipid homeostasis, whereas nonhepatic β-Kl regulates energy metabolism. We further demonstrate that in a condition with excessive cholesterol disposal, a robust compensatory mechanism maintains cholesterol levels but not triglyceride levels in mice. PMID:26514166

  20. Regulation of Liver Enriched Transcription Factors in Rat Hepatocytes Cultures on Collagen and EHS Sarcoma Matrices

    PubMed Central

    Borlak, Jürgen; Singh, Prafull Kumar; Rittelmeyer, Ina

    2015-01-01

    Liver-enriched transcription factors (LETF) play a crucial role in the control of liver-specific gene expression and for hepatocytes to retain their molecular and cellular functions complex interactions with extra cellular matrix (ECM) are required However, during cell isolation ECM interactions are disrupted and for hepatocytes to regain metabolic competency cells are cultured on ECM substrata. The regulation of LETFs in hepatocytes cultured on different ECM has not been studied in detail. We therefore compared two common sources of ECM and evaluated cellular morphology and hepatocyte differentiation by investigating DNA binding activity of LETFs at gene specific promoters and marker genes of hepatic metabolism. Furthermore, we studied testosterone metabolism and albumin synthesis to assess the metabolic competence of cell cultures. Despite significant difference in morphological appearance and except for HNF1β (p<0.001) most LETFs and several of their target genes did not differ in transcript expression after Bonferroni adjustment when cultured on collagen or Matrigel. Nonetheless, Western blotting revealed HNF1β, HNF3α, HNF3γ, HNF4α, HNF6 and the smaller subunits of C/EBPα and C/EBPβ to be more abundant on Matrigel cultured cells. Likewise, DNA binding activity of HNF3α, HNF3β, HNF4α, HNF6 and gene expression of hepatic lineage markers were increased on Matrigel cultured hepatocytes. To further investigate hepatic gene regulation, the effects of Aroclor 1254 treatment, e.g. a potent inducer of xenobiotic defense were studied in vivo and in vitro. The gene expression of C/EBP-α increased in rat liver and hepatocytes cultured on collagen and this treatment induced DNA binding activity of HNF4α, C/EBPα and C/EBPβ and gene expression of CYP1A1 and CYP1A2 in vivo and in vitro. Taken collectively, two sources of ECM greatly affected hepatocyte morphology, activity of liver enriched transcription factors, hepatic gene expression and metabolic competency

  1. Alterations of cell volume regulation in the development of hepatocyte necrosis.

    PubMed

    Carini, R; Autelli, R; Bellomo, G; Albano, E

    1999-04-10

    Intracellular Na+ accumulation has been shown to contribute to hepatocyte death caused by anoxia or oxidative stress. In this study we have investigated the mechanism by which Na+ overload can contribute to the development of cytotoxicity. ATP depletion in isolated hepatocytes exposed to menadione-induced oxidative stress or to KCN was followed by Na+ accumulation, loss of intracellular K+, and cell swelling. Hepatocyte swelling occurred in two phases: a small amplitude swelling (about 15% of the initial size) with preservation of plasma membrane integrity and a terminal large amplitude swelling associated with cell death. Inhibition of Na+ accumulation by the use of a Na+-free medium prevented K+ loss, cell swelling, and cytotoxicity. Conversely, blocking K+ efflux by the addition of BaCl2 did not influence Na+ increase and small amplitude swelling, but greatly stimulated large amplitude swelling and cytotoxicity. Menadione or KCN killing of hepatocytes was also enhanced by inducing cell swelling in an hypotonic medium. However, increasing the osmolarity of the incubation medium did not protect against large amplitude swelling and cytotoxicity, since stimulated Na+ accumulation and K+ efflux. Altogether these results indicate that the impairment of volume regulation in response to the osmotic load caused by Na+ accumulation is critical for the development of cell necrosis induced by mitochondrial inhibition or oxidative stress. PMID:10094834

  2. Growth Hormone-Regulated mRNAs and miRNAs in Chicken Hepatocytes

    PubMed Central

    Wang, Huijuan; Shao, Fang; Yu, JianFeng; Jiang, Honglin; Han, Yaoping; Gong, Daoqing; Gu, Zhiliang

    2014-01-01

    Growth hormone (GH) is a key regulatory factor in animal growth, development and metabolism. Based on the expression level of the GH receptor, the chicken liver is a major target organ of GH, but the biological effects of GH on the chicken liver are not fully understood. In this work we identified mRNAs and miRNAs that are regulated by GH in primary hepatocytes from female chickens through RNA-seq, and analyzed the functional relevance of these mRNAs and miRNAs through GO enrichment analysis and miRNA target prediction. A total of 164 mRNAs were found to be differentially expressed between GH-treated and control chicken hepatocytes, of which 112 were up-regulated and 52 were down-regulated by GH. A total of 225 chicken miRNAs were identified by the RNA-Seq analysis. Among these miRNAs 16 were up-regulated and 1 miRNA was down-regulated by GH. The GH-regulated mRNAs were mainly involved in growth and metabolism. Most of the GH-upregulated or GH-downregulated miRNAs were predicted to target the GH-downregulated or GH-upregulated mRNAs, respectively, involved in lipid metabolism. This study reveals that GH regulates the expression of many mRNAs involved in metabolism in female chicken hepatocytes, which suggests that GH plays an important role in regulating liver metabolism in female chickens. The results of this study also support the hypothesis that GH regulates lipid metabolism in chicken liver in part by regulating the expression of miRNAs that target the mRNAs involved in lipid metabolism. PMID:25386791

  3. A novel herbal formulation "LiverCare" differentially regulates primary rat hepatocyte and hepatocarcinoma cell proliferation in vitro.

    PubMed

    Vidyashankar, Satyakumar; Varma, Sandeep R; Azeemudin, Mohammed; Godavarthi, Ashok; Krishna, Nandakumar S; Patki, Pralhad Sadashiv

    2011-09-01

    Hepatocyte growth factor (HGF) plays an important role in hepatocyte proliferation. HGF expression is regulated by various signaling molecules and nuclear receptors. In the present study, LiverCare(®) (LC), a novel polyherbal formulation (The Himalaya Drug Company, Bangalore, India), was evaluated for its efficacy, using co-cultures of primary rat hepatocytes-non-parenchymal cells (NPCs) and human hepatocellular carcinoma cells (HepG2). The rate of primary hepatocyte co-culture proliferation was significantly and dose-dependently increased by LC as determined by [(3)H]thymidine incorporation into newly synthesized DNA and cell proliferation assay. LC also increased HGF expression in primary hepatocyte co-culture. Albumin and urea content remained constant during proliferation of hepatocyte co-cultures in the presence of LC with decreased activity of alanine aminotransferase. It is interesting that LC inhibited incorporation of [(3)H]thymidine into DNA in HepG2 cells. LC enhanced peroxisome proliferator-activated receptor-α expression during hepatocyte proliferation, whereas tumor necrosis factor-α expression remained unaffected. In conclusion, our study clearly showed that LC differentially regulates primary rat hepatocytes and human hepatocarcinoma cell proliferation. LC may be a promising candidate for treating degenerative liver diseases by enhancing liver regeneration. PMID:21812649

  4. The machine intelligence Hex project

    NASA Astrophysics Data System (ADS)

    Chalup, Stephan K.; Mellor, Drew; Rosamond, Fran

    2005-12-01

    Hex is a challenging strategy board game for two players. To enhance students’ progress in acquiring understanding and practical experience with complex machine intelligence and programming concepts we developed the Machine Intelligence Hex (MIHex) project. The associated undergraduate student assignment is about designing and implementing Hex players and evaluating them in an automated tournament of all programs developed by the class. This article surveys educational aspects of the MIHex project. Additionally, fundamental techniques for game programming as well as specific concepts for Hex board evaluation are reviewed. The MIHex game server and possibilities of tournament organisation are described. We summarise and discuss our experiences from running the MIHex project assignment over four consecutive years. The impact on student motivation and learning benefits are evaluated using questionnaires and interviews.

  5. Effect of glucagon on intracellular pH regulation in isolated rat hepatocyte couplets.

    PubMed Central

    Alvaro, D; Della Guardia, P; Bini, A; Gigliozzi, A; Furfaro, S; La Rosa, T; Piat, C; Capocaccia, L

    1995-01-01

    To elucidate mechanisms of glucagon-induced bicarbonate-rich choleresis, we investigated the effect of glucagon on ion transport processes involved in the regulation of intracellular pH (pHi) in isolated rat hepatocyte couplets. It was found that glucagon (200 nM), without influencing resting pHi, significantly stimulates the Cl-/HCO3- exchange activity. The effect of glucagon was associated with a sevenfold increase in cAMP levels in rat hepatocytes. The activity of the Cl-/HCO3- exchanger was also stimulated by DBcAMP + forskolin. The effect of glucagon on the Cl-/HCO3- exchange was individually blocked by two specific and selective inhibitors of protein kinase A, Rp-cAMPs (10 microM) and H-89 (30 microM), the latter having no influence on the glucagon-induced cAMP accumulation in isolated rat hepatocytes. The Cl- channel blocker, NPPB (10 microM), showed no effect on either the basal or the glucagon-stimulated Cl-/HCO3 exchange. In contrast, the protein kinase C agonist, PMA (10 microM), completely blocked the glucagon stimulation of the Cl-/HCO3- exchange; however, this effect was achieved through a significant inhibition of the glucagon-stimulated cAMP accumulation in rat hepatocytes. Colchicine pretreatment inhibited the basal as well as the glucagon-stimulated Cl-/HCO3- exchange activity. The Na+/H+ exchanger was unaffected by glucagon either at basal pHi or at acid pHi values. In contrast, glucagon, at basal pHi, stimulated the Na(+)-HCO3- symport. The main findings of this study indicate that glucagon, through the cAMP-dependent protein kinase A pathway, stimulates the activity of the Cl-/HCO3- exchanger in isolated rat hepatocyte couplets, a mechanism which could account for the in vivo induced bicarbonate-rich choleresis. Images PMID:7635959

  6. IL-6 modulates hepatocyte proliferation via induction of HGF/p21{sup cip1}: Regulation by SOCS3

    SciTech Connect

    Sun Rui; Jaruga, Barbara; Kulkarni, Shailin; Sun Haoyu; Gao Bin . E-mail: bgao@mail.nih.gov

    2005-12-30

    The precise role of IL-6 in liver regeneration and hepatocyte proliferation is controversial and the role of SOCS3 in liver regeneration remains unknown. Here we show that in vitro treatment with IL-6 inhibited primary mouse hepatocyte proliferation. IL-6 induced p21{sup cip1} protein expression in primary mouse hepatocytes. Disruption of the p21{sup cip1} gene abolished the inhibitory effect of IL-6 on cell proliferation. Co-culture with nonparenchymal liver cells diminished IL-6 inhibition of hepatocyte proliferation, which was likely due to IL-6 stimulation of nonparenchymal cells to produce HGF. Finally, IL-6 induced higher levels of p21{sup cip1} protein expression and a slightly stronger inhibition of cell proliferation in SOCS3{sup +/-} mouse hepatocytes compared to wild-type hepatocytes, while liver regeneration was enhanced and prolonged in SOCS3{sup +/-} mice. Our findings suggest that IL-6 directly inhibits hepatocyte proliferation via a p21{sup cip1}-dependent mechanism and indirectly enhances hepatocyte proliferation via stimulating nonparenchymal cells to produce HGF. SOCS3 negatively regulates liver regeneration.

  7. SUMOylation of HNF4α regulates protein stability and hepatocyte function.

    PubMed

    Zhou, Wenli; Hannoun, Zara; Jaffray, Ellis; Medine, Claire N; Black, James R; Greenhough, Sebastian; Zhu, Liang; Ross, James A; Forbes, Stuart; Wilmut, Ian; Iredale, John P; Hay, Ronald T; Hay, David C

    2012-08-01

    The coordination of signalling pathways within the cell is vital for normal human development and post-natal tissue homeostasis. Gene expression and function is therefore tightly controlled at a number of levels. We investigated the role that post-translational modifications play during human hepatocyte differentiation. In particular, we examined the role of the small ubiquitin-like modifier (SUMO) proteins in this process. We used a human embryonic stem cell (hESC)-based model of hepatocyte differentiation to follow changes in protein SUMOylation. Moreover, to confirm the results derived from our cell-based system, we performed in vitro conjugation assays to characterise SUMO modification of a key liver-enriched transcription factor, HNF4α. Our analyses indicate that SUMOylation plays an important role during hepatocellular differentiation and this is mediated, in part, through regulation of the stability of HNF4α in a ubiquitin-dependent manner. Our study provides a better understanding of SUMOylation during human hepatocyte differentiation and maturation. Moreover, we believe the results will stimulate interest in the differentiation and phenotypic regulation of other somatic cell types. PMID:22505616

  8. Prolyl hydroxylase-1 regulates hepatocyte apoptosis in an NF-κB-dependent manner.

    PubMed

    Fitzpatrick, Susan F; Fábián, Zsolt; Schaible, Bettina; Lenihan, Colin R; Schwarzl, Thomas; Rodriguez, Javier; Zheng, Xingnan; Li, Zongwei; Tambuwala, Murtaza M; Higgins, Desmond G; O'Meara, Yvonne; Slattery, Craig; Manresa, Mario C; Fraisl, Peter; Bruning, Ulrike; Baes, Myriam; Carmeliet, Peter; Doherty, Glen; von Kriegsheim, Alex; Cummins, Eoin P; Taylor, Cormac T

    2016-06-01

    Hepatocyte death is an important contributing factor in a number of diseases of the liver. PHD1 confers hypoxic sensitivity upon transcription factors including the hypoxia inducible factor (HIF) and nuclear factor-kappaB (NF-κB). Reduced PHD1 activity is linked to decreased apoptosis. Here, we investigated the underlying mechanism(s) in hepatocytes. Basal NF-κB activity was elevated in PHD1(-/-) hepatocytes compared to wild type controls. ChIP-seq analysis confirmed enhanced binding of NF-κB to chromatin in regions proximal to the promoters of genes involved in the regulation of apoptosis. Inhibition of NF-κB (but not knock-out of HIF-1 or HIF-2) reversed the anti-apoptotic effects of pharmacologic hydroxylase inhibition. We hypothesize that PHD1 inhibition leads to altered expression of NF-κB-dependent genes resulting in reduced apoptosis. This study provides new information relating to the possible mechanism of therapeutic action of hydroxylase inhibitors that has been reported in pre-clinical models of intestinal and hepatic disease. PMID:27130823

  9. Sulfated Oxysterol, 25HC3S, is a Potent Regulator of Lipid Metabolism in Human Hepatocytes

    PubMed Central

    Ren, Shunlin; Li, Xiaobo; Rodriguez-Agudo, Daniel; Gil, Gregorio; Hylemon, Phillip; Pandak, William M.

    2009-01-01

    Recently, a novel oxysterol, 5-cholesten-3β, 25-diol 3-sulfate (25HC3S) was identified in primary rat hepatocytes following overexpression of the cholesterol transport protein, StarD1. This oxysterol was also detected in human liver nuclei. In the present study, 25HC3S was chemically synthesized. Addition of 25HC3S (6 μM) to human hepatocytes markedly inhibited cholesterol biosynthesis. Quantitative RT-PCR and Western blot analysis showed that 25HC3S strongly decreased HMG-CoA reductase mRNA and protein levels. Coincidently, 25HC3S inhibited the activation of sterol regulatory element binding proteins (SREBPs), suggesting that inhibition of cholesterol biosynthesis occurred via blocking SREBP-1 activation, and subsequently inhibiting the expression of HMG CoA reductase. 25HC3S decreased SREBP-1 mRNA levels and inhibited the expression of target genes encoding acetyl CoA carboxylase-1 (ACC-1) and fatty acid synthase (FAS). In contract, 25-hydroxycholesterol increased SREBP1 and FAS mRNA levels in primary human hepatocytes. The results imply that 25HC3S is a potent regulator of SREBPs mediated lipid metabolism. PMID:17624300

  10. An HNF1α-regulated feedback circuit modulates hepatic fibrogenesis via the crosstalk between hepatocytes and hepatic stellate cells

    PubMed Central

    Qian, Hui; Deng, Xing; Huang, Zhao-Wei; Wei, Ji; Ding, Chen-Hong; Feng, Ren-Xin; Zeng, Xin; Chen, Yue-Xiang; Ding, Jin; Qiu, Lei; Hu, Zhen-Lin; Zhang, Xin; Wang, Hong-Yang; Zhang, Jun-Ping; Xie, Wei-Fen

    2015-01-01

    Hepatocytes are critical for the maintenance of liver homeostasis, but its involvement in hepatic fibrogenesis remains elusive. Hepatocyte nuclear factor 1α (HNF1α) is a liver-enriched transcription factor that plays a key role in hepatocyte function. Our previous study revealed a significant inhibitory effect of HNF1α on hepatocellular carcinoma. In this study, we report that the expression of HNF1α is significantly repressed in both human and rat fibrotic liver. Knockdown of HNF1α in the liver significantly aggravates hepatic fibrogenesis in either dimethylnitrosamine (DMN) or bile duct ligation (BDL) model in rats. In contrast, forced expression of HNF1α markedly alleviates hepatic fibrosis. HNF1α regulates the transcriptional expression of SH2 domain-containing phosphatase-1 (SHP-1) via directly binding to SHP-1 promoter in hepatocytes. Inhibition of SHP-1 expression abrogates the anti-fibrotic effect of HNF1α in DMN-treated rats. Moreover, HNF1α repression in primary hepatocytes leads to the activation of NF-κB and JAK/STAT pathways and initiates an inflammatory feedback circuit consisting of HNF1α, SHP-1, STAT3, p65, miR-21 and miR-146a, which sustains the deregulation of HNF1α in hepatocytes. More interestingly, a coordinated crosstalk between hepatocytes and hepatic stellate cells (HSCs) participates in this positive feedback circuit and facilitates the progression of hepatocellular damage. Our findings demonstrate that impaired hepatocytes play an active role in hepatic fibrogenesis. Early intervention of HNF1α-regulated inflammatory feedback loop in hepatocytes may have beneficial effects in the treatment of chronic liver diseases. PMID:26169608

  11. Regulation of drug transporter expression by oncostatin M in human hepatocytes.

    PubMed

    Le Vee, Marc; Jouan, Elodie; Stieger, Bruno; Lecureur, Valérie; Fardel, Olivier

    2011-08-01

    The cytokine oncostatin M (OSM) is a member of the interleukin (IL)-6 family, known to down-regulate expression of drug metabolizing cytochromes P-450 in human hepatocytes. The present study was designed to determine whether OSM may also impair expression of sinusoidal and canalicular drug transporters, which constitute important determinants of drug hepatic clearance. Exposure of primary human hepatocytes to OSM down-regulated mRNA levels of major sinusoidal solute carrier (SLC) influx transporters, including sodium-taurocholate co-transporting polypeptide (NTCP), organic anion transporting polypeptide (OATP) 1B1, OATP1B3, OATP2B1, organic cation transporter 1 and organic anion transporter 2. OSM also repressed mRNA expressions of ATP binding cassette (ABC) efflux transporters such as multidrug resistance protein (MRP) 2/ABCC2 and breast cancer resistance protein/ABCG2, without however impairing those of multidrug resistance gene 1/P-glycoprotein/ABCB1, MRP3/ABCC3, MRP4/ABCC4 and bile salt export pump/ABCB11. The cytokine concomitantly reduced NTCP, OATP1B1, OATP2B1 and ABCG2 protein expression and NTCP and OATP transport activities. OSM effects towards transporters were found to be dose-dependent and highly correlated with those of IL-6, but not with those of other inflammatory cytokines such as tumor necrosis factor-α or interferon-γ. In addition, OSM-mediated repression of some transporters such as NTCP, OATP1B1 and OATP2B1, was counteracted by knocking-down expression of the type II OSM receptor subunits through siRNA transfection. This OSM-mediated down-regulation of drug SLC transporters and ABCG2 in human hepatocytes may contribute to alterations of pharmacokinetics in patients suffering from diseases associated with increased production of OSM. PMID:21570956

  12. SRC-3 is required for CAR-regulated hepatocyte proliferation and drug metabolism

    PubMed Central

    Chen, Tenghui; Chen, Qiang; Xu, Yixiang; Zhou, Qiling; Zhu, Jingwei; Zhang, Hao; Wu, Qiao; Xu, Jianming; Yu, Chundong

    2011-01-01

    Background & Aims Nuclear receptors such as pregnane X receptor and constitutive androstane receptor (CAR) are important regulators of drug-metabolizing systems such as P450s enzymes and modulate xenobiotic metabolism as well as hepatocellular proliferation. Binding of CAR to NR response elements alone is not sufficient to activate gene expression. Here we investigate the role of steroid receptor coactivator (SRC) family members in CAR-mediated hepatocyte proliferation and drug metabolism. Methods The role of SRCs in CAR activation was assessed in cell-based transfection assays and protein-protein interaction assays. The in vivo role of SRCs in CAR-mediated hepatocyte proliferation and drug metabolism was examined by using mice deficient in SRCs. Results SRC-3 displayed the highest coactivating activity to CAR compared with SRC-1 and SRC-2 in a cell-based reporter assay. Knockout of SRC-3 in mice attenuated hepatic hyperplasia induced by a CAR agonist 1,4-bis-[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), which was associated with a reduced expression of c-Myc and Foxm-1. In contrast, knockout of SRC-1 or SRC-2 in mice did not affect TCPOBOP-induced hepatic hyperplasia. SRC-3-deficient mice were hypersensitive to zoxazolamine-induced paralysis, but were resistant to acetaminophen hepatotoxicity induced by TCPOBOP, whereas mutant mice deficient in SRC-1 or SRC-2 exhibited severe acetaminophen hepatotoxicity similar to wild-type controls. Accordingly, deficiency in SRC-3, but not SRC-1 or SRC-2, resulted in a reduced CAR-mediated expression of drug metabolism-related genes in the liver. Conclusions Our study demonstrates that SRC-3 is the predominant transcriptional coactivator among the three SRC family members for CAR activation to promote hepatocyte proliferation and drug metabolism. PMID:21827731

  13. PPAR{alpha} regulates the hepatotoxic biomarker alanine aminotransferase (ALT1) gene expression in human hepatocytes

    SciTech Connect

    Thulin, Petra; Rafter, Ingalill; Stockling, Kenneth; Tomkiewicz, Celine; Norjavaara, Ensio; Aggerbeck, Martine; Hellmold, Heike; Ehrenborg, Ewa; Andersson, Ulf; Cotgreave, Ian; Glinghammar, Bjoern

    2008-08-15

    In this work, we investigated a potential mechanism behind the observation of increased aminotransferase levels in a phase I clinical trial using a lipid-lowering drug, the peroxisome proliferator-activated receptor (PPAR) {alpha} agonist, AZD4619. In healthy volunteers treated with AZD4619, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were elevated without an increase in other markers for liver injury. These increases in serum aminotransferases have previously been reported in some patients receiving another PPAR{alpha} agonist, fenofibrate. In subsequent in vitro studies, we observed increased expression of ALT1 protein and mRNA in human hepatocytes after treatment with fenofibric acid. The PPAR effect on ALT1 expression was shown to act through a direct transcriptional mechanism involving at least one PPAR response element (PPRE) in the proximal ALT1 promoter, while no effect of fenofibrate and AZD4619 was observed on the ALT2 promoter. Binding of PPARs to the PPRE located at - 574 bp from the transcriptional start site was confirmed on both synthetic oligonucleotides and DNA in hepatocytes. These data show that intracellular ALT expression is regulated by PPAR agonists and that this mechanism might contribute to increased ALT activity in serum.

  14. Effects of adrenalectomy on the control and adrenergic regulation of cytosolic free calcium in hepatocytes

    SciTech Connect

    Freudenrich, C.C.

    1987-01-01

    The purpose of this study was to investigate the effects of adrenalectomy on the control and ..cap alpha..-adrenergic regulation of the concentration of cytosolic free calcium (Ca/sub i/) in hepatocytes. In hepatocytes isolated from adrenalectomized (adx) and sham-operated male rats 7-1 days after surgery, Ca/sub i/ at rest and in response to epinephrine (EPI) was measured with the calcium-sensitive photoprotein aequorin, /sup 45/Ca efflux was measured, and Ca/sup 2 +/ release from intracellular stores in response to inositol triphosphate (IP/sub 3/) was measured in saponin-permeabilized cells. Liver calmodulin content was also assayed by radioimmunoassay. It was found in adx rats that the resting Ca/sub i/ was elevated, the rise in Ca/sub i/ during EPI stimulation was reduced at physiological EPI concentrations, and the rise in calcium efflux evoked by EPI was reduced. Furthermore, the slope of the relationship between Ca/sub i/ and calcium efflux was reduced 60% in adx. Adx did not alter the characteristics of Ca/sup 2 +/ release from intracellular calcium pools in response to IP/sub 3/ in permeabilized cells. Finally, the liver calmodulin contents were not significantly different between the 2 groups.

  15. Hepatocyte nuclear factor-1β: A regulator of kidney development and cystogenesis

    PubMed Central

    Singh, V.; Singla, S. K.; Jha, V.; Puri, V.; Puri, S.

    2015-01-01

    The understanding of the genomics of the renal tissue has gathered a considerable interest and is making rapid progress. The molecular mechanisms as well as the precise function of the associated molecular components toward renal pathophysiology have recently been realized. For the cystic kidney disease, the regulation of gene expression affecting epithelial cells proliferation, apoptosis as well as process of differentiation/de-differentiation represent key molecular targets. For the cystic disorders, molecular targets have been identified, which besides lending heterogeneity to cysts may also provide tools to unravel their functional importance to understand the renal tissue homeostasis. This review focuses on providing comprehensive information about the transcriptional regulatory role of hepatocyte nuclear factor-1β, a homeoprotein, as well as its interacting partners in renal tissue development and pathophysiology. PMID:25838642

  16. Extracellular matrix controls tubulin monomer levels in hepatocytes by regulating protein turnover

    NASA Technical Reports Server (NTRS)

    Mooney, D. J.; Hansen, L. K.; Langer, R.; Vacanti, J. P.; Ingber, D. E.

    1994-01-01

    Cells have evolved an autoregulatory mechanism to dampen variations in the concentration of tubulin monomer that is available to polymerize into microtubules (MTs), a process that is known as tubulin autoregulation. However, thermodynamic analysis of MT polymerization predicts that the concentration of free tubulin monomer must vary if MTs are to remain stable under different mechanical loads that result from changes in cell adhesion to the extracellular matrix (ECM). To determine how these seemingly contradictory regulatory mechanisms coexist in cells, we measured changes in the masses of tubulin monomer and polymer that resulted from altering cell-ECM contacts. Primary rat hepatocytes were cultured in chemically defined medium on bacteriological petri dishes that were precoated with different densities of laminin (LM). Increasing the LM density from low to high (1-1000 ng/cm2), promoted cell spreading (average projected cell area increased from 1200 to 6000 microns2) and resulted in formation of a greatly extended MT network. Nevertheless, the steady-state mass of tubulin polymer was similar at 48 h, regardless of cell shape or ECM density. In contrast, round hepatocytes on low LM contained a threefold higher mass of tubulin monomer when compared with spread cells on high LM. Furthermore, similar results were obtained whether LM, fibronectin, or type I collagen were used for cell attachment. Tubulin autoregulation appeared to function normally in these cells because tubulin mRNA levels and protein synthetic rates were greatly depressed in round cells that contained the highest level of free tubulin monomer. However, the rate of tubulin protein degradation slowed, causing the tubulin half-life to increase from approximately 24 to 55 h as the LM density was lowered from high to low and cell rounding was promoted. These results indicate that the set-point for the tubulin monomer mass in hepatocytes can be regulated by altering the density of ECM contacts and

  17. Hepatitis C virus suppresses Hepatocyte Nuclear Factor 4 alpha, a key regulator of hepatocellular carcinoma.

    PubMed

    Vallianou, Ioanna; Dafou, Dimitra; Vassilaki, Niki; Mavromara, Penelope; Hadzopoulou-Cladaras, Margarita

    2016-09-01

    Hepatitis C Virus (HCV) infection presents with a disturbed lipid profile and can evolve to hepatic steatosis and hepatocellular carcinoma (HCC). Hepatocyte Nuclear Factor 4 alpha (HNF4α) is the most abundant transcription factor in the liver, a key regulator of hepatic lipid metabolism and a critical determinant of Epithelial to Mesenchymal Transition and hepatic development. We have previously shown that transient inhibition of HNF4α initiates transformation of immortalized hepatocytes through a feedback loop consisting of miR-24, IL6 receptor (IL6R), STAT3, miR-124 and miR-629, suggesting a central role of HNF4α in HCC. However, the role of HNF4α in Hepatitis C Virus (HCV)-related hepatocarcinoma has not been evaluated and remains controversial. In this study, we provide strong evidence suggesting that HCV downregulates HNF4α expression at both transcriptional and translational levels. The observed decrease of HNF4α expression correlated with the downregulation of its downstream targets, HNF1α and MTP. Ectopic overexpression of HCV proteins also exhibited an inhibitory effect on HNF4α levels. The inhibition of HNF4α expression by HCV appeared to be mediated at transcriptional level as HCV proteins suppressed HNF4α gene promoter activity. HCV also up-regulated IL6R, activated STAT3 protein phosphorylation and altered the expression of acute phase genes. Furthermore, as HCV triggered the loss of HNF4α a consequent change of miR-24, miR-629 or miR-124 was observed. Our findings demonstrated that HCV-related HCC could be mediated through HNF4α-microRNA deregulation implying a possible role of HNF4α in HCV hepatocarcinogenesis. HCV inhibition of HNF4α could be sustained to promote HCC. PMID:27477312

  18. MicroRNA-194 Regulates Hepatocytic Differentiation of Progenitor Cells by Targeting YAP1

    PubMed Central

    Jung, Kwang Hwa; McCarthy, Ryan L.; Zhou, Chong; Uprety, Nadima; Barton, Michelle Craig; Beretta, Laura

    2015-01-01

    MicroRNA expression profiling in human liver progenitor cells following hepatocytic differentiation identified miR-122 and miR-194 as the microRNAs most strongly upregulated during hepatocytic differentiation of progenitor cells. MiR-194 was also highly upregulated following hepatocytic differentiation of human embryonic stem cells (hESCs). Overexpression of miR-194 in progenitor cells accelerated their differentiation into hepatocytes, as measured by morphological features such as canaliculi and expression of hepatocytic markers. Overexpression of miR-194 in hESCs induced their spontaneous differentiation, a phenotype accompanied with accelerated loss of the pluripotent factors OCT4 and NANOG and decrease in mesoderm marker HAND1 expression. We then identified YAP1 as a direct target of miR-194. Inhibition of YAP1 strongly induced hepatocytic differentiation of progenitor cells and YAP1 over expression reversed the miR-194-induced hepatocytic differentiation of progenitor cells. In conclusion, we identified miR-194 as a potent inducer of hepatocytic differentiation of progenitor cells and further identified YAP1 as a mediator of miR-194's effects on hepatocytic differentiation and liver progenitor cell fate. PMID:26731713

  19. Hepatocyte nuclear factor-4alpha and bile acids regulate human concentrative nucleoside transporter-1 gene expression.

    PubMed

    Klein, Kerstin; Kullak-Ublick, Gerd A; Wagner, Martin; Trauner, Michael; Eloranta, Jyrki J

    2009-04-01

    The concentrative nucleoside transporter-1 (CNT1) is a member of the solute carrier 28 (SLC28) gene family and is expressed in the liver, intestine, and kidneys. CNT1 mediates the uptake of naturally occurring pyrimidine nucleosides, but also nucleoside analogs used in anticancer and antiviral therapy. Thus expression levels of CNT1 may affect the pharmacokinetics of these drugs and the outcome of drug therapy. Because little is known about the transcriptional regulation of human CNT1 gene expression, we have characterized the CNT1 promoter with respect to DNA response elements and their binding factors. The transcriptional start site of the CNT1 gene was determined by 5'-RACE. In silico analysis revealed the existence of three putative binding sites for the nuclear receptor hepatocyte nuclear factor-4alpha (HNF-4alpha) within the CNT1 promoter. A luciferase reporter gene construct containing the CNT1 promoter region was transactivated by HNF-4alpha in human cell lines derived from the liver, intestine, and kidneys. Consistent with this, we showed in electromobility shift assays that HNF-4alpha specifically binds to two conserved direct repeat-1 motifs within the proximal CNT1 promoter. In cotransfection experiments, the transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator-1alpha further increased, whereas the bile acid-inducible corepressor small heterodimer partner reduced, HNF-4alpha-dependent CNT1 promoter activity. Consistent with the latter phenomenon, CNT1 mRNA expression levels were suppressed in primary human hepatocytes upon bile acid treatment. Supporting the physiological relevance and species conservation of this effect, ileal Cnt1 mRNA expression was decreased upon bile acid feeding and increased upon bile duct ligation in mice. PMID:19228884

  20. The regulation of cytoskeletal and liver-specific gene expression during liver regeneration and primary hepatocyte culture

    SciTech Connect

    Robinson, G.S.

    1989-01-01

    The focus of this dissertation is to determine what role(s) the extracellular matrix and expression of certain cytoskeletal genes play in the regulation of hepatocyte growth and the maintenance of a differential state. The expression of several cytoskeletal and liver-specific genes was examined during liver regeneration and in hepatocyte cultures maintained in a hormonally-defined, serum-free medium and plated on two different matrices: rat tail collagen and the EHS matrix. During liver regeneration and in hepatocytes cultured on rat tail collagen, there was a dramatic increase in tubulin mRNA levels coincident with but not linked to DNA synthesis. The message levels for other cytoskeletal genes similarly increased, while a decrease was observed in the mRNA levels of the liver-specific genes, serum albumin and alpha{sub 1} inhibitor III. Hepatocytes cultured on the EHS matrix resulted in the maintenance of low levels of cytoskeletal gene expression and high levels of liver-specific gene expression, similar to that observed in the normal liver. Results from subcellar fractionation and two-dimensional gel electrophoresis of {sup 35}S-labelled proteins paralleled the results seen at the mRNA level. Preliminary work suggests that microtubule organization may play a role in the expression of the liver-specific genes which encode secreted proteins. These studies, which compare hepatocytes cultured on collagen or the EHS matrix gel, reveal that both cell-cell and cell-matrix interactions play a major role in the maintenance of the differential phenotype in hepatocytes.

  1. Expression of Hex during feather bud development.

    PubMed

    Obinata, Akiko; Akimoto, Yoshihiro

    2005-01-01

    We studied proline-rich divergent homeobox gene Hex/Prh expression in the dorsal skin of chick embryo during feather bud development. Hex mRNA expression was first observed in the dorsolateral ectoderm and mesenchyme at 5 days, then in the epithelium and the dermis of the dorsal skin before placode (primordium of feather bud) formation and then was restricted to the placode and the dermis under the placode. Afterward, Hex expression was seen in the epidermis and the dermis of the posterior region of short bud. In accordance with Hex mRNA expression in the placode, Hex protein was observed in the epidermis as well as in the dermis of the placode. Immunoelectron microscopic study indicated that the protein located both in the nuclei and cytoplasm of the epidermis and the dermis at the short bud stage. The Wnt signaling pathway plays an essential role in the early inductive events in hair (Wnt3a and 7a) and feather (Wnt7a) follicles. The pattern of Hex expression in the epidermis was similar to that of Wnt7a, while little, if any, expression of Wnt7a was detected in the dermis under the placode or the dermis of the short bud compared with that of Hex, suggesting that Hex plays an important role in the initiation of feather morphogenesis. PMID:16172986

  2. Modeling wildlife populations with HexSim

    EPA Science Inventory

    HexSim is a framework for constructing spatially-explicit, individual-based computer models designed for simulating terrestrial wildlife population dynamics and interactions. HexSim is useful for a broad set of modeling applications including population viability analysis for on...

  3. The Role of Hepatocyte Hemojuvelin in the Regulation of Bone Morphogenic Protein-6 and Hepcidin Expression in Vivo*

    PubMed Central

    Zhang, An-Sheng; Gao, Junwei; Koeberl, Dwight D.; Enns, Caroline A.

    2010-01-01

    Both hemojuvelin (HJV) and bone morphogenic protein-6 (BMP6) are essential for hepcidin expression. Hepcidin is the key peptide hormone in iron homeostasis, and is secreted predominantly by hepatocytes. HJV expression is detected in hepatocytes, as well as in skeletal and heart muscle. HJV binds BMP6 and increases hepcidin expression presumably by acting as a BMP co-receptor. We characterized the role of hepatocyte HJV in the regulation of BMP6 and hepcidin expression. In HJV-null (Hjv−/−) mice that have severe iron overload and marked suppression of hepcidin expression, we detected 4-fold higher hepatic BMP6 mRNA than in wild-type counterparts. These results indicate that Hjv−/− mice do not lack BMP6. Furthermore, iron depletion in Hjv−/− mice decreased hepatic BMP6 mRNA. Expression of HJV in hepatocytes of Hjv−/− mice using an AAV2/8 vector, increased hepatic hepcidin mRNA by 65-fold and phosphorylated Smad1/5/8 in the liver by about 2.5-fold. However, no significant change in BMP6 mRNA was detected in either the liver or the small intestine of these animals. Our results revealed a close correlation of hepatic BMP6 mRNA expression with hepatic iron-loading. Together, our data indicate that the regulation of hepatic BMP6 expression by iron is independent of HJV, and that expression of HJV in hepatocytes plays an essential role in hepcidin expression by potentiating the BMP6-mediated signaling. PMID:20363739

  4. The role of hepatocyte hemojuvelin in the regulation of bone morphogenic protein-6 and hepcidin expression in vivo.

    PubMed

    Zhang, An-Sheng; Gao, Junwei; Koeberl, Dwight D; Enns, Caroline A

    2010-05-28

    Both hemojuvelin (HJV) and bone morphogenic protein-6 (BMP6) are essential for hepcidin expression. Hepcidin is the key peptide hormone in iron homeostasis, and is secreted predominantly by hepatocytes. HJV expression is detected in hepatocytes, as well as in skeletal and heart muscle. HJV binds BMP6 and increases hepcidin expression presumably by acting as a BMP co-receptor. We characterized the role of hepatocyte HJV in the regulation of BMP6 and hepcidin expression. In HJV-null (Hjv(-/-)) mice that have severe iron overload and marked suppression of hepcidin expression, we detected 4-fold higher hepatic BMP6 mRNA than in wild-type counterparts. These results indicate that Hjv(-/-) mice do not lack BMP6. Furthermore, iron depletion in Hjv(-/-) mice decreased hepatic BMP6 mRNA. Expression of HJV in hepatocytes of Hjv(-/-) mice using an AAV2/8 vector, increased hepatic hepcidin mRNA by 65-fold and phosphorylated Smad1/5/8 in the liver by about 2.5-fold. However, no significant change in BMP6 mRNA was detected in either the liver or the small intestine of these animals. Our results revealed a close correlation of hepatic BMP6 mRNA expression with hepatic iron-loading. Together, our data indicate that the regulation of hepatic BMP6 expression by iron is independent of HJV, and that expression of HJV in hepatocytes plays an essential role in hepcidin expression by potentiating the BMP6-mediated signaling. PMID:20363739

  5. Acid sphingomyelinase regulates glucose and lipid metabolism in hepatocytes through AKT activation and AMP-activated protein kinase suppression

    PubMed Central

    Osawa, Yosuke; Seki, Ekihiro; Kodama, Yuzo; Suetsugu, Atsushi; Miura, Kouichi; Adachi, Masayuki; Ito, Hiroyasu; Shiratori, Yoshimune; Banno, Yoshiko; Olefsky, Jerrold M.; Nagaki, Masahito; Moriwaki, Hisataka; Brenner, David A.; Seishima, Mitsuru

    2011-01-01

    Acid sphingomyelinase (ASM) regulates the homeostasis of sphingolipids, including ceramides and sphingosine-1-phosphate (S1P). Because sphingolipids regulate AKT activation, we investigated the role of ASM in hepatic glucose and lipid metabolism. Initially, we overexpressed ASM in the livers of wild-type and diabetic db/db mice by adenovirus vector (Ad5ASM). In these mice, glucose tolerance was improved, and glycogen and lipid accumulation in the liver were increased. Using primary cultured hepatocytes, we confirmed that ASM increased glucose uptake, glycogen deposition, and lipid accumulation through activation of AKT and glycogen synthase kinase-3β. In addition, ASM induced up-regulation of glucose transporter 2 accompanied by suppression of AMP-activated protein kinase (AMPK) phosphorylation. Loss of sphingosine kinase-1 (SphK1) diminished ASM-mediated AKT phosphorylation, but exogenous S1P induced AKT activation in hepatocytes. In contrast, SphK1 deficiency did not affect AMPK activation. These results suggest that the SphK/S1P pathway is required for ASM-mediated AKT activation but not for AMPK inactivation. Finally, we found that treatment with high-dose glucose increased glycogen deposition and lipid accumulation in wild-type hepatocytes but not in ASM−/− cells. This result is consistent with glucose intolerance in ASM−/− mice. In conclusion, ASM modulates AKT activation and AMPK inactivation, thus regulating glucose and lipid metabolism in the liver.—Osawa, Y., Seki, E., Kodama, Y., Suetsugu, A., Miura, K., Adachi, M., Ito, H., Shiratori, Y., Banno, Y., Olefsky, J. M., Nagaki, M., Moriwaki, H., Brenner, D. A., Seishima, M. Acid sphingomyelinase regulates glucose and lipid metabolism in hepatocytes through AKT activation and AMP-activated protein kinase suppression. PMID:21163859

  6. Involvement of hepatocyte nuclear factor 1 in the regulation of the UDP-glucuronosyltransferase 1A7 (UGT1A7) gene in rat hepatocytes.

    PubMed

    Metz, R P; Auyeung, D J; Kessler, F K; Ritter, J K

    2000-08-01

    UDP-glucuronosyltransferase 1A7 (UGT1A7) is a major UGT contributing to the glucuronidation of xenobiotic phenols in rats. Its expression in rat liver is tightly regulated, with low constitutive and high inducible expression in response to aryl hydrocarbon receptor ligands and oltipraz. Previously, we reported the absence of 3-methylcholanthrene- or oltipraz-responsive elements in the 1.6-kbp region flanking the UGT1A7 promoter. However, potential binding sites were noted for several liver-enriched transcription factors. Here we show that deletion of the hepatic nuclear factor (HNF)3, HNF4, and CCAAT-enhancer binding protein-like binding sites had no effect on the expression of a UGT1A7 reporter plasmid, p(-965/+56)1A7-Luc, in primary rat hepatocytes. The full activity of the promoter was contained in the region between bases -157 and +76. Two sites of binding by rat liver nuclear proteins were detected in this region by DNase footprinting. PR-1 corresponded to the HNF1-like binding site between bases -52 and -38, whereas PR-2 was located between -30 to -6. Gel retardation studies supported the presence of HNF1alpha in the PR-1 DNA-liver nuclear protein complex. Mutation of PR-1 inhibited binding in the gel shift assay, prevented activation by overexpressed HNF1 in human embryonic kidney cells, and reduced by >80% the maximal luciferase activities expressed from basal and 3-methylcholanthrene-responsive UGT1A7 gene reporter constructs in primary rat hepatocytes. These data provide evidence for an important stimulatory role of HNF1 in promoting UGT1A7 gene expression in rat liver. PMID:10908299

  7. Regulation of bile acid biosynthesis by hepatocyte nuclear factor 4α

    PubMed Central

    Inoue, Yusuke; Yu, Ai-Ming; Yim, Sun Hee; Ma, Xiaochao; Krausz, Kristopher W.; Inoue, Junko; Xiang, Charlie C.; Brownstein, Michael J.; Eggertsen, Gösta; Björkhem, Ingemar

    2005-01-01

    Hepatocyte nuclear factor 4α (HNF4α) regulates many genes that are preferentially expressed in liver. Mice lacking hepatic expression of HNF4α, HNF4αΔL, exhibited markedly elevated levels of serum bile acids compared to HNF4α-floxed mice, HNF4αF/F. The expression of genes involved in the hydroxylation and side chain β-oxidation of cholesterol including oxysterol 7α-hydroxylase (CYP7B1), sterol 12α-hydroxylase (CYP8B1), and sterol carrier protein x (SCPx) was markedly decreased in HNF4αΔL mice. Cholesterol 7α-hydroxylase (CYP7A1) mRNA and protein were diminished only during the dark cycle in HNF4αΔL mice, whereas expression in the light cycle was not different between and HNF4αΔL and HNF4αF/F mice. Since CYP8B1 expression was reduced in HNF4αΔL mice, it was studied in more detail. In agreement with the mRNA levels, CYP8B1 enzyme activity was absent in HNF4αΔL mice. An HNF4α binding site was found in the mouse Cyp8b1 promoter that was able to direct HNF4α-dependent transcription. Surprisingly, cholic acid-derived BAs, produced as a result of CYP8B1 activity, were still observed in the serum and gallbladder of these mice. These studies reveal that HNF4α plays a central role in BA homeostasis by regulation of genes involved in BA biosynthesis including hydroxylation and side chain β-oxidation of cholesterol in vivo. PMID:16264197

  8. Heat Shock Protein 27 is down-regulated in Ballooned Hepatocytes of Patients with Nonalcoholic Steatohepatitis (NASH)

    PubMed Central

    Sookoian, Silvia; Castaño, Gustavo O.; Scian, Romina; San Martino, Julio; Pirola, Carlos J.

    2016-01-01

    Ballooning degeneration (BD) of hepatocytes is a distinguishing histological feature associated with the progression of nonalcoholic fatty liver disease (NAFLD). Under the assumption that NAFLD severity is associated with metabolic-stress we explored the hypothesis that heat shock 27 kDa protein 1 (HSP27), a protein chaperone involved in stress resistance and cytoskeletal-remodeling, might be deregulated in ballooned hepatocytes. We observed that fasting plasma glucose (fpG) (p = 0.00002), total cholesterol (p = 0.02) and triglycerides (p = 0.01) levels, and female sex (p = 0.01) were significantly associated with the presence of BD. A logistic regression model showed that BD was independently associated with fpG (p = 0.002); OR per unit of glucose concentration 1.05, 95% confidence interval 1.02–1.09. Furthermore, BD was associated with a significant 2.24-fold decrease in the expression level of HSP27-mRNA in comparison with absence of ballooning, p = 0.002. Ballooned hepatocytes showed very low HSP27 immunoreactivity compared with hepatocyes without ballooning (p = 0.009); HSP27 immunoreactivity was inversely correlated with fpG levels (R: −0.49, p = 0.01). In conclusion, BD is associated with down-regulation of liver HSP27 gene and protein expression, suggesting that ballooned hepatocytes fail to ensure a robust physiological response to metabolic-induced stress. PMID:26935030

  9. Heat Shock Protein 27 is down-regulated in Ballooned Hepatocytes of Patients with Nonalcoholic Steatohepatitis (NASH).

    PubMed

    Sookoian, Silvia; Castaño, Gustavo O; Scian, Romina; San Martino, Julio; Pirola, Carlos J

    2016-01-01

    Ballooning degeneration (BD) of hepatocytes is a distinguishing histological feature associated with the progression of nonalcoholic fatty liver disease (NAFLD). Under the assumption that NAFLD severity is associated with metabolic-stress we explored the hypothesis that heat shock 27 kDa protein 1 (HSP27), a protein chaperone involved in stress resistance and cytoskeletal-remodeling, might be deregulated in ballooned hepatocytes. We observed that fasting plasma glucose (fpG) (p = 0.00002), total cholesterol (p = 0.02) and triglycerides (p = 0.01) levels, and female sex (p = 0.01) were significantly associated with the presence of BD. A logistic regression model showed that BD was independently associated with fpG (p = 0.002); OR per unit of glucose concentration 1.05, 95% confidence interval 1.02-1.09. Furthermore, BD was associated with a significant 2.24-fold decrease in the expression level of HSP27-mRNA in comparison with absence of ballooning, p = 0.002. Ballooned hepatocytes showed very low HSP27 immunoreactivity compared with hepatocyes without ballooning (p = 0.009); HSP27 immunoreactivity was inversely correlated with fpG levels (R: -0.49, p = 0.01). In conclusion, BD is associated with down-regulation of liver HSP27 gene and protein expression, suggesting that ballooned hepatocytes fail to ensure a robust physiological response to metabolic-induced stress. PMID:26935030

  10. HexR Controls Glucose-Responsive Genes and Central Carbon Metabolism in Neisseria meningitidis

    PubMed Central

    Antunes, Ana; Golfieri, Giacomo; Ferlicca, Francesca; Giuliani, Marzia M.; Scarlato, Vincenzo

    2015-01-01

    ABSTRACT Neisseria meningitidis, an exclusively human pathogen and the leading cause of bacterial meningitis, must adapt to different host niches during human infection. N. meningitidis can utilize a restricted range of carbon sources, including lactate, glucose, and pyruvate, whose concentrations vary in host niches. Microarray analysis of N. meningitidis grown in a chemically defined medium in the presence or absence of glucose allowed us to identify genes regulated by carbon source availability. Most such genes are implicated in energy metabolism and transport, and some are implicated in virulence. In particular, genes involved in glucose catabolism were upregulated, whereas genes involved in the tricarboxylic acid cycle were downregulated. Several genes encoding surface-exposed proteins, including the MafA adhesins and Neisseria surface protein A, were upregulated in the presence of glucose. Our microarray analysis led to the identification of a glucose-responsive hexR-like transcriptional regulator that controls genes of the central carbon metabolism of N. meningitidis in response to glucose. We characterized the HexR regulon and showed that the hexR gene is accountable for some of the glucose-responsive regulation; in vitro assays with the purified protein showed that HexR binds to the promoters of the central metabolic operons of the bacterium. Based on DNA sequence alignment of the target sites, we propose a 17-bp pseudopalindromic consensus HexR binding motif. Furthermore, N. meningitidis strains lacking hexR expression were deficient in establishing successful bacteremia in an infant rat model of infection, indicating the importance of this regulator for the survival of this pathogen in vivo. IMPORTANCE Neisseria meningitidis grows on a limited range of nutrients during infection. We analyzed the gene expression of N. meningitidis in response to glucose, the main energy source available in human blood, and we found that glucose regulates many genes

  11. Suppression in PHLPP2 induction by morin promotes Nrf2-regulated cellular defenses against oxidative injury to primary rat hepatocytes

    PubMed Central

    Rizvi, Fatima; Mathur, Alpana; Krishna, Shagun; Siddiqi, Mohammad Imran; Kakkar, Poonam

    2015-01-01

    Recent advances indicate a possible role of phytochemicals as modulatory factors in signaling pathways. We have previously demonstrated PHLPP2-mediated suppression of Nrf2 responses during oxidant attack. The present study was designed to explore Nrf2-potentiating mechanism of morin, a flavonol, via its possible role in intervening PHLPP2-regulated Akt/GSK3β/Fyn kinase axis. Efficacy of morin was evaluated against oxidative stress-mediated damage to primary hepatocytes by tert-butyl hydroperoxide (tBHP) and acetaminophen. The anti-cytotoxic effects of morin were found to be a consequence of fortification of Nrf2-regulated antioxidant defenses since morin failed to sustain activities of redox enzyme in Nrf2 silenced hepatocytes. Morin promoted Nrf2 stability and its nuclear retention by possibly modulating PHLPP2 activity which subdues cellular Nrf2 responses by activating Fyn kinase. Pull-down assay using morin-conjugated beads indicated the binding affinity of morin towards PHLPP2. Molecular docking also revealed the propensity of morin to occupy the active site of PHLPP2 enzyme. Thus, dietary phytochemical morin was observed to counteract oxidant-induced hepatocellular damage by promoting Nrf2-regulated transcriptional induction. The findings support the novel role of morin in potentiating Nrf2 responses by limiting PHLPP2 and hence Fyn kinase activation. Therefore, morin may be exploited in developing novel therapeutic strategy aimed at enhancing Nrf2 responses. PMID:26513344

  12. Hepatocyte NADPH Oxidase 4 Regulates Stress Signaling, Fibrosis, and Insulin Sensitivity During Development of Steatohepatitis in Mice

    PubMed Central

    Bettaieb, Ahmed; Jiang, Joy X.; Sasaki, Yu; Chao, Tzu-I; Kiss, Zsofia; Chen, Xiangling; Tian, Jijing; Katsuyama, Masato; Yabe-Nishimura, Chihiro; Xi, Yannan; Szyndralewiez, Cedric; Schröder, Kathrin; Shah, Ajay; Brandes, Ralph P.; Haj, Fawaz G.; Török, Natalie J.

    2015-01-01

    Background & Aims Reactive oxidative species (ROS) are believed to be involved in the progression of non-alcoholic steatohepatitis (NASH). However, little is known about the sources of ROS in hepatocytes or their role in disease progression. We studied the effects of NADPH oxidase 4 (NOX4) in liver tissues from patients with NASH and mice with steatohepatitis. Methods Liver biopsy samples were obtained from 5 patients with NASH, as well as 4 patients with simple steatosis and 5 patients without steatosis (controls) from the University of California, Davis Cancer Center Biorepository. Mice with hepatocyte-specific deletion of NOX4 (NOX4hepKO) and NOX4floxp+/+ C57BL/6 mice (controls) were given fast food diets (supplemented with high-fructose corn syrup) or choline-deficient L-amino acid-defined to induce steatohepatitis, or control diets, for 20 weeks. A separate group of mice were given the NOX4 inhibitor (GKT137831). Liver tissues were collected and immunoblot analyses were performed determine levels of NOX4, markers of inflammation and fibrosis, double-stranded RNA-activated protein kinase (PKR), and phospho-eIF-2alpha kinase (PERK)-mediated stress signaling pathways. We performed hyperinsulinemic-euglycemic clamp studies and immunoprecipitation analyses to determine the oxidation and phosphatase activity of PP1C. Results Levels of NOX4 were increased in patients with NASH, compared with controls. Hepatocyte-specific deletion of NOX4 reduced oxidative stress, lipid peroxidation, and liver fibrosis in mice with diet-induced steatohepatitis. A small molecule inhibitor of NOX4 reduced liver inflammation and fibrosis and increased insulin sensitivity in mice with diet-induced steatohepatitis. In primary hepatocytes, NOX4 reduced the activity of the phosphatase PP1C, prolonging activation of PKR and PERK-mediated stress signaling. Mice with hepatocyte-specific deletion of NOX4 and mice given GKT137831had increased insulin sensitivity. Conclusion NOX4 regulates

  13. Coactivator PGC-1{alpha} regulates the fasting inducible xenobiotic-metabolizing enzyme CYP2A5 in mouse primary hepatocytes

    SciTech Connect

    Arpiainen, Satu; Jaervenpaeae, Sanna-Mari; Manninen, Aki; Viitala, Pirkko; Lang, Matti A.; Pelkonen, Olavi; Hakkola, Jukka

    2008-10-01

    The nutritional state of organisms and energy balance related diseases such as diabetes regulate the metabolism of xenobiotics such as drugs, toxins and carcinogens. However, the mechanisms behind this regulation are mostly unknown. The xenobiotic-metabolizing cytochrome P450 (CYP) 2A5 enzyme has been shown to be induced by fasting and by glucagon and cyclic AMP (cAMP), which mediate numerous fasting responses. Peroxisome proliferator-activated receptor {gamma} coactivator (PGC)-1{alpha} triggers many of the important hepatic fasting effects in response to elevated cAMP levels. In the present study, we were able to show that cAMP causes a coordinated induction of PGC-1{alpha} and CYP2A5 mRNAs in murine primary hepatocytes. Furthermore, the elevation of the PGC-1{alpha} expression level by adenovirus mediated gene transfer increased CYP2A5 transcription. Co-transfection of Cyp2a5 5' promoter constructs with the PGC-1{alpha} expression vector demonstrated that PGC-1{alpha} is able to activate Cyp2a5 transcription through the hepatocyte nuclear factor (HNF)-4{alpha} response element in the proximal promoter of the Cyp2a5 gene. Chromatin immunoprecipitation assays showed that PGC-1{alpha} binds, together with HNF-4{alpha}, to the same region at the Cyp2a5 proximal promoter. In conclusion, PGC-1{alpha} mediates the expression of CYP2A5 induced by cAMP in mouse hepatocytes through coactivation of transcription factor HNF-4{alpha}. This strongly suggests that PGC-1{alpha} is the major factor mediating the fasting response of CYP2A5.

  14. Effects of adrenalectomy on the alpha-adrenergic regulation of cytosolic free calcium in hepatocytes

    SciTech Connect

    Freudenrich, C.C.; Borle, A.B.

    1988-06-25

    We have previously published that bilateral adrenalectomy in the rat reduces the Ca2+-mediated alpha-adrenergic activation of hepatic glycogenolysis, while it increases the cellular calcium content of hepatocytes. In the experiments presented here, the concentration of cytosolic free calcium (Ca2+i) at rest and in response to epinephrine was measured in aequorin-loaded hepatocytes isolated from sham and adrenalectomized male rats. We found that in adrenalectomized rats the resting Ca2+i was elevated, the rise in Ca2+i evoked by epinephrine was reduced, and the rise in /sup 45/Ca efflux that follows such stimulation was depressed. Furthermore, the slope of the relationship between Ca2+i and calcium efflux was decreased 60% in adrenalectomized. Adrenalectomy did not change Ca2+ release from intracellular calcium pools in response to IP3 in saponin-permeabilized hepatocytes. The EC50 for inositol 1,4,5-triphosphate and the maximal Ca2+ released were similar in both sham and adrenalectomized animals. Finally, the liver calmodulin content determined by radioimmunoassay was not significantly different between sham and adrenalectomized rats. These results suggest that 1) adrenalectomy reduces calcium efflux from the hepatocyte, probably by an effect on the plasma membrane (Ca2+-Mg2+)-ATPase-dependent Ca2+ pump and thus alters cellular calcium homeostasis; 2) adrenalectomy decreases the rise in Ca2+i in response to epinephrine; 3) this decreased rise in Ca2+i is not due to defects in the intracellular Ca2+ storage and mobilization processes; and 4) the effects of adrenalectomy on cellular calcium metabolism and on alpha-adrenergic activation of glycogenolysis are not caused by a reduction in soluble calmodulin.

  15. Transcriptional Regulation of CYP2B6 Expression by Hepatocyte Nuclear Factor 3β in Human Liver Cells

    PubMed Central

    Li, Linhao; Li, Daochuan; Heyward, Scott; Wang, Hongbing

    2016-01-01

    CYP2B6 plays an increasingly important role in xenobiotic metabolism and detoxification. The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) have been established as predominant regulators for the inductive expression of CYP2B6 gene in human liver. However, there are dramatic interindividual variabilities in CYP2B6 expression that cannot be fully explained by the CAR/PXR-based modulation alone. Here, we show that expression level of CYP2B6 was correlated with that of hepatocyte nuclear factor 3β (HNF3β) in human primary hepatocytes prepared from 35 liver donors. Utilizing recombinant virus-mediated overexpression or knockdown of HNF3β in HepG2 cells, as well as constructs containing serial deletion and site-directed mutation of HNF3β binding motifs in CYP2B6 luciferase reporter assays, we demonstrated that the presence or lack of HNF3β expression markedly correlated with CYP2B6 gene expression and its promoter activity. Novel enhancer modules of HNF3β located upstream of the CYP2B6 gene transcription start site were identified and functionally validated as key elements governing HNF3β-mediated CYP2B6 expression. Chromatin immunoprecipitation assays in human primary hepatocytes and surface plasmon resonance binding affinity experiments confirmed the essential role of these enhancers in the recruitment of HNF3β to the promoter of CYP2B6 gene. Overall, these findings indicate that HNF3β represents a new liver enriched transcription factor that is involved in the transcription of CYP2B6 gene and contributes to the large interindividual variations of CYP2B6 expression in human population. PMID:26930610

  16. Idh1 protects murine hepatocytes from endotoxin-induced oxidative stress by regulating the intracellular NADP(+)/NADPH ratio.

    PubMed

    Itsumi, M; Inoue, S; Elia, A J; Murakami, K; Sasaki, M; Lind, E F; Brenner, D; Harris, I S; Chio, I I C; Afzal, S; Cairns, R A; Cescon, D W; Elford, A R; Ye, J; Lang, P A; Li, W Y; Wakeham, A; Duncan, G S; Haight, J; You-Ten, A; Snow, B; Yamamoto, K; Ohashi, P S; Mak, T W

    2015-11-01

    Isocitrate dehydrogenase-1 (Idh1) is an important metabolic enzyme that produces NADPH by converting isocitrate to α-ketoglutarate. Idh1 is known to reduce reactive oxygen species (ROS) induced in cells by treatment with lipopolysaccharide (LPS) in vitro. Here, we used Idh1-deficient knockout (Idh1 KO) mice to investigate the role of Idh1 in antioxidant defense in vivo. Idh1 KO mice showed heightened susceptibility to death induced by LPS and exhibited increased serum levels of inflammatory cytokines such as tumor necrosis factor-α and interleukin-6. The serum of LPS-injected Idh1 KO mice also contained elevated levels of AST, a marker of inflammatory liver damage. Furthermore, after LPS injection, livers of Idh1 KO mice showed histological evidence of elevated oxidative DNA damage compared with livers of wild-type (WT) mice. Idh1 KO livers showed a faster and more pronounced oxidative stress than WT livers. In line with that, Idh1 KO hepatocytes showed higher ROS levels and an increase in the NADP(+)/NADPH ratio when compared with hepatocytes isolated from WT mice. These results suggest that Idh1 has a physiological function in protecting cells from oxidative stress by regulating the intracellular NADP(+)/NADPH ratio. Our findings suggest that stimulation of Idh1 activity may be an effective therapeutic strategy for reducing oxidative stress during inflammatory responses, including the early stages of septic shock. PMID:25882048

  17. Hex Chrome Free Coatings for Electronics Overview

    NASA Technical Reports Server (NTRS)

    Kessel, Kurt

    2013-01-01

    The overall objective of the Hex Chrome Free Coatings for Electronics project is to evaluate and test pretreatment coating systems not containing hexavalent chrome in avionics and electronics housing applications. This objective will be accomplished by testing strong performing coating systems from prior NASA and DoD testing or new coating systems as determined by the stakeholders.

  18. Potency of individual bile acids to regulate bile acid synthesis and transport genes in primary human hepatocyte cultures.

    PubMed

    Liu, Jie; Lu, Hong; Lu, Yuan-Fu; Lei, Xiaohong; Cui, Julia Yue; Ellis, Ewa; Strom, Stephen C; Klaassen, Curtis D

    2014-10-01

    Bile acids (BAs) are known to regulate their own homeostasis, but the potency of individual bile acids is not known. This study examined the effects of cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA) and ursodeoxycholic acid (UDCA) on expression of BA synthesis and transport genes in human primary hepatocyte cultures. Hepatocytes were treated with the individual BAs at 10, 30, and 100μM for 48 h, and RNA was extracted for real-time PCR analysis. For the classic pathway of BA synthesis, BAs except for UDCA markedly suppressed CYP7A1 (70-95%), the rate-limiting enzyme of bile acid synthesis, but only moderately (35%) down-regulated CYP8B1 at a high concentration of 100μM. BAs had minimal effects on mRNA of two enzymes of the alternative pathway of BA synthesis, namely CYP27A1 and CYP7B1. BAs increased the two major target genes of the farnesoid X receptor (FXR), namely the small heterodimer partner (SHP) by fourfold, and markedly induced fibroblast growth factor 19 (FGF19) over 100-fold. The BA uptake transporter Na(+)-taurocholate co-transporting polypeptide was unaffected, whereas the efflux transporter bile salt export pump was increased 15-fold and OSTα/β were increased 10-100-fold by BAs. The expression of the organic anion transporting polypeptide 1B3 (OATP1B3; sixfold), ATP-binding cassette (ABC) transporter G5 (ABCG5; sixfold), multidrug associated protein-2 (MRP2; twofold), and MRP3 (threefold) were also increased, albeit to lesser degrees. In general, CDCA was the most potent and effective BA in regulating these genes important for BA homeostasis, whereas DCA and CA were intermediate, LCA the least, and UDCA ineffective. PMID:25055961

  19. Na(+)-Ca sup 2+ exchange in cultured rat hepatocytes: Evidence against a role in cytosolic Ca sup 2+ regulation or signaling

    SciTech Connect

    Lidofsky, S.D.; Xie, M.H.; Scharschmidt, B.F. )

    1990-07-01

    Plasma membrane Na(+)-Ca2+ exchange contributes importantly to the regulation of cytosolic Ca2+ concentration ((Ca2+)i) in excitable cells. Despite extensive study in excitable tissues, the role of this transporter in the regulation of (Ca2+)i in hepatocytes is unknown, and conflicting information has been reported regarding the presence of Na(+)-Ca2+ exchange in hepatocyte plasma membrane vesicles. We have therefore assessed the role of Na(+)-dependent Ca2+ transport in the regulation of (Ca2+)i in rat hepatocytes in primary culture under basal conditions and after exposure to vasopressin, a hormone that elevates (Ca2+)i. Ca2+ efflux, measured using 45Ca, did not differ in the presence or absence of extracellular Na+, either under basal conditions or in response to vasopressin. (Ca2+)i, measured using the Ca2(+)-sensitive dye fura-2, was not altered by transient or prolonged exposure to Na(+)-free media or by exposure to ouabain in concentrations sufficient to produce a five-fold elevation in intracellular Na+ concentration. The (Ca2+)i response to vasopressin was also unaffected by Na+ removal or ouabain. By contrast, in cultured rat cardiac myocytes, cells that possess Na(+)-Ca2+ exchange, transient or prolonged Na+ removal as well as ouabain exposure produced greater than fivefold increases in (Ca2+)i compared with controls. We conclude that Na(+)-Ca2+ exchange does not contribute to the regulation of (Ca2+)i in hepatocytes.

  20. Fucoidan protects hepatocytes from apoptosis and inhibits invasion of hepatocellular carcinoma by up-regulating p42/44 MAPK-dependent NDRG-1/CAP43

    PubMed Central

    Cho, Yuri; Yoon, Jung-Hwan; Yoo, Jeong-ju; Lee, Minjong; Lee, Dong Hyeon; Cho, Eun Ju; Lee, Jeong-Hoon; Yu, Su Jong; Kim, Yoon Jun; Kim, Chung Yong

    2015-01-01

    Fucoidan is a traditional Chinese medicine suggested to possess anti-tumor effects. In this study the anti-metastatic effects of fucoidan were investigated in vitro in human hepatocellular carcinoma (HCC) cells (Huh-7 and SNU-761) under normoxic and hypoxic conditions and in vivo using a distant liver metastasis model involving injection of MH134 cells into spleen via the portal vein. Its ability to protect hepatocytes against bile acid (BA)-induced apoptosis was investigated in primary hepatocytes. Fucoidan was found to suppress the invasion of HCC cells through up-regulation of p42/44 MAPK-dependent NDRG-1/CAP43 and partly, under normoxic conditions, through up-regulation of p42/44 MAPK-dependent VMP-1 expression. It also significantly decreased liver metastasis in vivo. As regards its hepatoprotective effect, fucoidan decreased BA-induced hepatocyte apoptosis as shown by the attenuation of caspase-8, and -7 cleavages and suppression of the mobilization of caspase-8 and Fas associated death domain (FADD) into the death-inducing signaling complex. In summary, fucoidan displays inhibitory effects on proliferation of HCC cells and protective effects on hepatocytes. The results suggest fucoidan is a potent suppressor of tumor invasion with hepatoprotective effects. PMID:26713269

  1. Transcriptional Up-Regulation of APE1/Ref-1 in Hepatic Tumor: Role in Hepatocytes Resistance to Oxidative Stress and Apoptosis

    PubMed Central

    Di Maso, Vittorio; Mediavilla, María Gabriela; Vascotto, Carlo; Lupo, Francesco; Baccarani, Umberto; Avellini, Claudio; Tell, Gianluca; Tiribelli, Claudio; Crocè, Lory Saveria

    2015-01-01

    Objective Human Hepatocellular Carcinoma (HCC) is the fifth most frequent neoplasm worldwide and the most serious complication of long-standing chronic liver diseases (CLD). Its development is associated with chronic inflammation and sustained oxidative stress. Deregulation of apurinic apyrimidinic endonuclease 1/redox effector factor 1 (APE1/Ref-1), a master regulator of cellular response to oxidative stress, has been associated with poor prognosis in several cancers including HCC. Design In the present study we investigated the APE1/Ref-1 mRNA levels in cirrhotic and HCC tissues obtained during HCC resection. The possible protective role of APE1/Ref-1 against oxidative stress and apoptosis was evaluated in vitro in immortalized human hepatocytes (IHH) over-expressing APE1/Ref-1. Results APE1/Ref-1 was up-regulated in HCC, regulation occurring at the transcriptional level. APE1/Ref-1 mRNA content increased with the progression of liver disease with the transcriptional up-regulation present in cirrhosis significantly increased in HCC. The up-regulation was higher in the less differentiated cancers. In vitro, over-expression of APE1/Ref-1 in normal hepatocytes conferred cell protection against oxidative stress and it was associated with BAX inhibition and escape from apoptosis. Conclusion APE1/Ref-1 is up-regulated in HCC and this over-expression correlates with cancer aggressiveness. The up-regulation occurs at the transcriptional level and it is present in the earliest phases of hepatocarcinogenesis. The APE-1/Ref-1 over-expression is associated with hepatocyte survival and inhibits BAX activation and apoptosis. These data suggest a possible role of APE1/Ref-1 over-expression both in hepatocyte survival and HCC development calling attention to this molecule as a promising marker for HCC diagnosis and treatment. PMID:26624999

  2. Internal regulation of ATP turnover, glycolysis and oxidative phosphorylation in rat hepatocytes.

    PubMed

    Ainscow, E K; Brand, M D

    1999-12-01

    Previously [Ainscow, E.K. & Brand, M.D. (1999) Eur. J. Biochem. 263, 671-685], top-down control analysis was used to describe the control pattern of energy metabolism in rat hepatocytes. The system was divided into nine reaction blocks (glycogen breakdown, glucose release, glycolysis, lactate production, NADH oxidation, pyruvate oxidation, mitochondrial proton leak, mitochondrial phosphorylation and ATP consumption) linked by five intermediates (intracellular glucose 6-phosphate, pyruvate and ATP levels, cytoplasmic NADH/NAD ratio and mitochondrial membrane potential). The kinetic responses (elasticities) of reaction blocks to intermediates were determined and used to calculate control coefficients. In the present paper, these elasticities and control coefficients are used to quantify the internal regulatory pathways within the cell. Flux control coefficients were partitioned to give partial flux control coefficients. These describe how strongly one block of reactions controls the flux through another via its effects on the concentration of a particular intermediate. Most flux control coefficients were the sum of positive and negative partial effects acting through different intermediates; these partial effects could be large compared to the final control strength. An important result was the breakdown of the way ATP consumption controlled respiration: changes in ATP level were more important than changes in mitochondrial membrane potential in stimulating oxygen consumption when ATP consumption increased. The partial internal response coefficients to changes in each intermediate were also calculated; they describe how steady state concentrations of intermediates are maintained. Increases in mitochondrial membrane potential were opposed mostly by decreased supply, whereas increases in glucose-6-phosphate, NADH/NAD and pyruvate were opposed mostly by increased consumption. Increases in ATP were opposed significantly by both decreased supply and increased consumption

  3. Hepatocyte nuclear factor-3 alpha (HNF-3{alpha}) negatively regulates androgen receptor transactivation in prostate cancer cells

    SciTech Connect

    Lee, Hyun Joo; Hwang, Miok; Chattopadhyay, Soma; Choi, Hueng-Sik; Lee, Keesook

    2008-03-07

    The androgen receptor (AR) is involved in the development and progression of prostate cancers. However, the mechanisms by which this occurs remain incompletely understood. In previous reports, hepatocyte nuclear factor-3{alpha} (HNF-3{alpha}) has been shown to be expressed in the epithelia of the prostate gland, and has been determined to regulate the transcription of prostate-specific genes. In this study, we report that HNF-3{alpha} functions as a novel corepressor of AR in prostatic cells. HNF-3{alpha} represses AR transactivation on target promoters containing the androgen response element (ARE) in a dose-dependent manner. HNF-3{alpha} interacts physically with AR, and negatively regulates AR transactivation via competition with AR coactivators, including GRIP1. Furthermore, HNF-3{alpha} overexpression reduces the androgen-induced expression of prostate-specific antigen (PSA) in LNCaP cells. Taken together, our findings indicate that HNF-3{alpha} is a novel corepressor of AR, and predict its effects on the proliferation of prostate cancer cells.

  4. Non-esterified fatty acids activate the AMP-activated protein kinase signaling pathway to regulate lipid metabolism in bovine hepatocytes.

    PubMed

    Li, Xinwei; Li, Xiaobing; Chen, Hui; Lei, Liancheng; Liu, Juxiong; Guan, Yuan; Liu, Zhaoxi; Zhang, Liang; Yang, Wentao; Zhao, Chenxu; Fu, Shixin; Li, Peng; Liu, Guowen; Wang, Zhe

    2013-01-01

    Non-esterified fatty acids (NEFAs) act as signaling molecules involved in regulating genes expression to modulate lipid metabolism. However, the regulation mechanism of NEFAs on lipid metabolism in dairy cows is unclear. The AMP-activated protein kinase (AMPK) signaling pathway plays a key role in regulating hepatic lipid metabolism. In vitro, bovine hepatocytes were cultured and treated with different concentrations of NEFAs and AMPKα inhibitors (BML-275). NEFAs increased AMPKα phosphorylation through up-regulating the protein levels of liver kinase B1. Activated AMPKα increased the expression and transcriptional activity of peroxisome proliferator-activated receptor α (PPARα). NEFAs also directly activate the PPARα independent of AMPKα. Activated PPARα increased the lipolytic genes expression to increase lipid oxidation. Furthermore, activated AMPKα inhibited the expression and transcriptional activity of the sterol regulatory element-binding protein 1c and carbohydrate responsive element-binding protein, which reduced the expression of lipogenic genes, thereby decreasing lipid synthesis. Activated AMPKα phosphorylated and inhibited acetyl-CoA carboxylase and increased carnitine palmitoyltransferase-1 activity, which increased lipid oxidation. Consequently, the triglyceride content in the NEFAs-treated hepatocytes was significantly decreased. These results indicate that NEFAs activate the AMPKα signaling pathway to increase lipid oxidation and decrease lipid synthesis in hepatocytes, which in turn, generates more ATP to relieve the negative energy balance in transition dairy cows. PMID:23690240

  5. Transcription Factor Hepatocyte Nuclear Factor-1β Regulates Renal Cholesterol Metabolism.

    PubMed

    Aboudehen, Karam; Kim, Min Soo; Mitsche, Matthew; Garland, Kristina; Anderson, Norma; Noureddine, Lama; Pontoglio, Marco; Patel, Vishal; Xie, Yang; DeBose-Boyd, Russell; Igarashi, Peter

    2016-08-01

    HNF-1β is a tissue-specific transcription factor that is expressed in the kidney and other epithelial organs. Humans with mutations in HNF-1β develop kidney cysts, and HNF-1β regulates the transcription of several cystic disease genes. However, the complete spectrum of HNF-1β-regulated genes and pathways is not known. Here, using chromatin immunoprecipitation/next generation sequencing and gene expression profiling, we identified 1545 protein-coding genes that are directly regulated by HNF-1β in murine kidney epithelial cells. Pathway analysis predicted that HNF-1β regulates cholesterol metabolism. Expression of dominant negative mutant HNF-1β or kidney-specific inactivation of HNF-1β decreased the expression of genes that are essential for cholesterol synthesis, including sterol regulatory element binding factor 2 (Srebf2) and 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr). HNF-1β mutant cells also expressed lower levels of cholesterol biosynthetic intermediates and had a lower rate of cholesterol synthesis than control cells. Additionally, depletion of cholesterol in the culture medium mitigated the inhibitory effects of mutant HNF-1β on the proteins encoded by Srebf2 and Hmgcr, and HNF-1β directly controlled the renal epithelial expression of proprotein convertase subtilisin-like kexin type 9, a key regulator of cholesterol uptake. These findings reveal a novel role of HNF-1β in a transcriptional network that regulates intrarenal cholesterol metabolism. PMID:26712526

  6. Hepatocyte growth factor regulates the TGF-β1-induced proliferation, differentiation and secretory function of cardiac fibroblasts

    PubMed Central

    YI, XIN; LI, XIAOYAN; ZHOU, YANLI; REN, SHAN; WAN, WEIGUO; FENG, GAOKE; JIANG, XUEJUN

    2014-01-01

    Cardiac fibroblast (CF) proliferation and transformation into myofibroblasts play important roles in cardiac fibrosis during pathological myocardial remodeling. In this study, we demonstrate that hepatocyte growth factor (HGF), an antifibrotic factor in the process of pulmonary, renal and liver fibrosis, is a negative regulator of cardiac fibroblast transformation in response to transforming growth factor-β1 (TGF-β1). HGF expression levels were significantly reduced in the CFs following treatment with 5 ng/ml TGF-β1 for 48 h. The overexpression of HGF suppressed the proliferation, transformation and the secretory function of the CFs following treatment with TGF-β1, as indicated by the attenuated expression levels of α-smooth muscle actin (α-SMA) and collagen I and III, whereas the knockdown of HGF had the opposite effect. Mechanistically, we identified that the phosphorylation of c-Met, Akt and total protein of TGIF was significantly inhibited by the knockdown of HGF, but was significantly enhanced by HGF overexpression. Collectively, these results indicate that HGF activates the c-Met-Akt-TGIF signaling pathway, inhibiting CF proliferation and transformation in response to TGF-β1 stimulation. PMID:24840640

  7. p65 down-regulates DEPTOR expression in response to LPS stimulation in hepatocytes.

    PubMed

    Yu, Xiaoling; Jin, Dan; Yu, An; Sun, Jun; Chen, Xiaodong; Yang, Zaiqing

    2016-09-01

    DEPTOR, a novel endogenous inhibitor of mTOR, plays an important role in regulating the inflammatory response in vascular endothelial cells (ECs) and in mouse skeletal muscle. However, the regulatory mechanism of DEPTOR transcription and its effects on liver inflammation are unknown presently. Here we reported the role of DEPTOR in regulating inflammatory response in mouse liver-derived Hepa1-6 cells and in a mouse model with LPS-induced hepatic inflammation. The results revealed that DEPTOR over-expression in Hepa1-6 liver cells increased the mRNA levels of the pro-inflammatory cytokines interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1). Contrasting results were observed in Hepa1-6 cells with DEPTOR interference. Treatment Hepa1-6 cells with rapamycin, a specific inhibitor of mTORC1, increased MCP-1 mRNA, but have no significant effect on IL-6 mRNA. DEPTOR expression was down-regulated in Hepa1-6 cells with the treatment of inflammatory stimuli LPS or the over-expression of p65/NF-κB, a key inflammatory transcription factor. NF-κB antagonist (PDTC) and inhibitor (IκBα) blocked the effect of LPS on DEPTOR expression. The study in vivo showed that DEPTOR mRNA and protein were significantly reduced in a mouse model with LPS-induced hepatic inflammation, which was accompanied by a concurrent activation of the mTOR signaling pathway. Further, the transcriptional regulation of DEPTOR was explored, which revealed that DEPTOR promoter activity was significantly down-regulated by NF-κB. The progressive deletions and mutations demonstrated that the NF-κB binding motif situated at -145/-127 region is an essential component required for the DEPTOR promoter activity. Chromatin immunoprecipitation (ChIP) assays determined that p65 can directly interact with the DEPTOR promoter DNA. Those results indicate DEPTOR regulates liver inflammation at least partially via mTORC1 pathway, and is down-regulated by LPS through p65. PMID:27179948

  8. Hepatocyte Transplantation

    PubMed Central

    Mitry, Ragai R; Hughes, Robin D; Dhawan, Anil

    2011-01-01

    Hepatocyte transplantation (HTx) has been developed for use in liver-based metabolic disorders and in acute liver failure. Worldwide, there are around 80 patients that have been transplanted with hepatocytes. Almost all reported studies prove feasibility and safety of the procedure with short- to medium-term success. Availability of good quality hepatocytes (HCs) is the main limiting factor, and therefore alternative sources of cells such as stem cells are being investigated. Other limiting factors include cell engraftment, survival, and function of transplanted cells. It remains to be seen if progress in HTx research can overcome these hurdles leading to the wider use of the technique as an alternative to liver transplantation in the future. PMID:25755322

  9. Hepatocyte growth factor regulated tyrosine kinase substrate in the peripheral development and function of B-cells

    SciTech Connect

    Nagata, Takayuki; Murata, Kazuko; Murata, Ryo; Sun, Shu-lan; Saito, Yutaro; Yamaga, Shuhei; Tanaka, Nobuyuki; Tamai, Keiichi; Moriya, Kunihiko; Kasai, Noriyuki; Sugamura, Kazuo; Ishii, Naoto

    2014-01-10

    Highlights: •ESCRT-0 protein regulates the development of peripheral B-cells. •BCR expression on cell surface should be controlled by the endosomal-sorting system. •Hrs plays important roles in responsiveness to Ag stimulation in B lymphocytes. -- Abstract: Hepatocyte growth factor (HGF)-regulated tyrosine kinase substrate (Hrs) is a vesicular sorting protein that functions as one of the endosomal-sorting proteins required for transport (ESCRT). Hrs, which binds to ubiquitinated proteins through its ubiquitin-interacting motif (UIM), contributes to the lysosomal transport and degradation of ubiquitinated membrane proteins. However, little is known about the relationship between B-cell functions and ESCRT proteins in vivo. Here we examined the immunological roles of Hrs in B-cell development and functions using B-cell-specific Hrs-deficient (Hrs{sup flox/flox};mb1{sup cre/+}:Hrs-cKO) mice, which were generated using a cre-LoxP recombination system. Hrs deficiency in B-cells significantly reduced T-cell-dependent antibody production in vivo and impaired the proliferation of B-cells treated in vitro with an anti-IgM monoclonal antibody but not with LPS. Although early development of B-cells in the bone marrow was normal in Hrs-cKO mice, there was a significant decrease in the number of the peripheral transitional B-cells and marginal zone B-cells in the spleen of Hrs-cKO mice. These results indicate that Hrs plays important roles during peripheral development and physiological functions of B lymphocytes.

  10. Regulation of polyunsaturated fatty acid biosynthesis by seaweed fucoxanthin and its metabolite in cultured hepatocytes.

    PubMed

    Aki, Tsunehiro; Yamamoto, Masaya; Takahashi, Toshiaki; Tomita, Kohki; Toyoura, Rieko; Iwashita, Kazuhiro; Kawamoto, Seiji; Hosokawa, Masashi; Miyashita, Kazuo; Ono, Kazuhisa

    2014-02-01

    The effects of a seaweed carotenoid, fucoxanthin, and its physiological metabolite, fucoxanthinol, on the biosynthesis of polyunsaturated fatty acids (PUFA) were investigated using cultured rat hepatoma BRL-3A. The metabolism of α-linolenic acid (18:3n-3) was suppressed by the addition of these carotenoids, resulting in a decrease in the content of eicosapentaenoic acid (20:5n-3), which suggested a down-regulation of metabolic enzymes such as fatty acid desaturase and elongase. An increase in the content of docosahexaenoic acid (22:6n-3), as observed in previous studies in vivo, might be a buffering action to maintain the membrane fluidity. The suppressive effect of fucoxanthinol on ∆6 fatty acid desaturase was not at the level of gene expression but due to specific modifications of the protein via a ubiquitin-proteasome system. A proteomic analysis revealed several factors such as phosphatidylethanolamine-binding protein that might be involved in the observed action of fucoxanthin. These findings will contribute to studies on the elucidation of the precise molecular mechanisms underlying the regulation of PUFA biosynthesis by fucoxanthin. PMID:24174374

  11. Intracellular pH regulation in rainbow trout (Oncorhynchus mykiss) hepatocytes: the activity of sodium/proton exchange is oxygen-dependent.

    PubMed

    Tuominen, A; Rissanen, E; Bogdanova, A; Nikinmaa, M

    2003-06-01

    We studied pH regulation in freshly isolated rainbow trout hepatocytes using microspectrofluorometry with the fluorescent dye BCECF. In accordance with earlier data on rainbow trout hepatocytes, ion substitution (N-methyl D-glucamine for sodium and gluconate for chloride) and transport inhibitor [10 microM M methyl isobutyl amiloride (MIA) to inhibit sodium/proton exchange and 100 microM DIDS to inhibit bicarbonate transport] studies in either Hepes-buffered or bicarbonate/carbon dioxide-buffered media (extracellular pH 7.6) indicated a role for sodium/proton exchange, sodium-dependent bicarbonate transport, and sodium-independent anion exchange in the regulation of hepatocyte pH. In Hepes-buffered medium, the activity of the sodium/proton exchanger (i.e. proton extrusion inhibited by MIA) was greater at 1% than at 21% oxygen. The oxygen dependency of the sodium/proton exchange is not caused by hydroxyl radicals, which appear to mediate the oxygen sensitivity of potassium-chloride cotransport in erythrocytes. PMID:12820008

  12. Keratin impact on PKCδ- and ASMase-mediated regulation of hepatocyte lipid raft size - implication for FasR-associated apoptosis.

    PubMed

    Gilbert, Stéphane; Loranger, Anne; Omary, M Bishr; Marceau, Normand

    2016-09-01

    Keratins are epithelial cell intermediate filament (IF) proteins that are expressed as pairs in a cell-differentiation-regulated manner. Hepatocytes express the keratin 8 and 18 pair (denoted K8/K18) of IFs, and a loss of K8 or K18, as in K8-null mice, leads to degradation of the keratin partner. We have previously reported that a K8/K18 loss in hepatocytes leads to altered cell surface lipid raft distribution and more efficient Fas receptor (FasR, also known as TNFRSF6)-mediated apoptosis. We demonstrate here that the absence of K8 or transgenic expression of the K8 G62C mutant in mouse hepatocytes reduces lipid raft size. Mechanistically, we find that the lipid raft size is dependent on acid sphingomyelinase (ASMase, also known as SMPD1) enzyme activity, which is reduced in absence of K8/K18. Notably, the reduction of ASMase activity appears to be caused by a less efficient redistribution of surface membrane PKCδ toward lysosomes. Moreover, we delineate the lipid raft volume range that is required for an optimal FasR-mediated apoptosis. Hence, K8/K18-dependent PKCδ- and ASMase-mediated modulation of lipid raft size can explain the more prominent FasR-mediated signaling resulting from K8/K18 loss. The fine-tuning of ASMase-mediated regulation of lipid rafts might provide a therapeutic target for death-receptor-related liver diseases. PMID:27422101

  13. Integrin-linked kinase (ILK) modulates wound healing through regulation of hepatocyte growth factor (HGF)

    SciTech Connect

    Serrano, Isabel; Diez-Marques, Maria L.; Rodriguez-Puyol, Manuel; Herrero-Fresneda, Inmaculada; Garcia del Moral, Raimundo; Dedhar, Shoukat; Ruiz-Torres, Maria P.; Rodriguez-Puyol, Diego

    2012-11-15

    Integrin-linked kinase (ILK) is an intracellular effector of cell-matrix interactions and regulates many cellular processes, including growth, proliferation, survival, differentiation, migration, invasion and angiogenesis. The present work analyzes the role of ILK in wound healing in adult animals using a conditional knock-out of the ILK gene generated with the tamoxifen-inducible Cre-lox system (CRE-LOX mice). Results show that ILK deficiency leads to retarded wound closure in skin. Intracellular mechanisms involved in this process were analyzed in cultured mouse embryonic fibroblast (MEF) isolated from CRE-LOX mice and revealed that wounding promotes rapid activation of phosphatidylinositol 3-kinase (PI3K) and ILK. Knockdown of ILK resulted in a retarded wound closure due to a decrease in cellular proliferation and loss of HGF protein expression during the healing process, in vitro and in vivo. Alterations in cell proliferation and wound closure in ILK-deficient MEF or mice could be rescued by exogenous administration of human HGF. These data demonstrate, for the first time, that the activation of PI3K and ILK after skin wounding are critical for HGF-dependent tissue repair and wound healing. -- Highlights: Black-Right-Pointing-Pointer ILK deletion results in decreased HGF expression and delayed scratch wound repair. Black-Right-Pointing-Pointer PI3K/ILK/AKT pathway signals through HGF to regulate wound healing. Black-Right-Pointing-Pointer An ILK-dependent increase in HGF expression is responsible for wound healing in vivo. Black-Right-Pointing-Pointer ILK-KO mice are used to confirm the requirement for ILK function in wound healing. Black-Right-Pointing-Pointer Human HGF treatment restores delayed wound closure in vitro and in vivo.

  14. Induction of mitochondrial biogenesis and respiration is associated with mTOR regulation in hepatocytes of rats treated with the pan-PPAR activator tetradecylthioacetic acid (TTA)

    SciTech Connect

    Hagland, Hanne R.; Nilsson, Linn I.H.; Burri, Lena; Nikolaisen, Julie; Berge, Rolf K.; Tronstad, Karl J.

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer We investigated mechanisms of mitochondrial regulation in rat hepatocytes. Black-Right-Pointing-Pointer Tetradecylthioacetic acid (TTA) was employed to activate mitochondrial oxidation. Black-Right-Pointing-Pointer Mitochondrial biogenesis and respiration were induced. Black-Right-Pointing-Pointer It was confirmed that PPAR target genes were induced. Black-Right-Pointing-Pointer The mechanism involved activation mTOR. -- Abstract: The hypolipidemic effect of peroxisome proliferator-activated receptor (PPAR) activators has been explained by increasing mitochondrial fatty acid oxidation, as observed in livers of rats treated with the pan-PPAR activator tetradecylthioacetic acid (TTA). PPAR-activation does, however, not fully explain the metabolic adaptations observed in hepatocytes after treatment with TTA. We therefore characterized the mitochondrial effects, and linked this to signalling by the metabolic sensor, the mammalian target of rapamycin (mTOR). In hepatocytes isolated from TTA-treated rats, the changes in cellular content and morphology were consistent with hypertrophy. This was associated with induction of multiple mitochondrial biomarkers, including mitochondrial DNA, citrate synthase and mRNAs of mitochondrial proteins. Transcription analysis further confirmed activation of PPAR{alpha}-associated genes, in addition to genes related to mitochondrial biogenesis and function. Analysis of mitochondrial respiration revealed that the capacity of both electron transport and oxidative phosphorylation were increased. These effects coincided with activation of the stress related factor, ERK1/2, and mTOR. The protein level and phosphorylation of the downstream mTOR actors eIF4G and 4E-BP1 were induced. In summary, TTA increases mitochondrial respiration by inducing hypertrophy and mitochondrial biogenesis in rat hepatocytes, via adaptive regulation of PPARs as well as mTOR.

  15. The five glucose-6-phosphatase paralogous genes are differentially regulated by insulin alone or combined with high level of amino acids and/or glucose in trout hepatocytes.

    PubMed

    Lucie, Marandel; Weiwei, Dai; Stéphane, Panserat; Sandrine, Skiba-Cassy

    2016-04-01

    A recent analysis of the newly sequenced rainbow trout (Oncorhynchus mykiss) genome suggested that duplicated gluconeogenic g6pc paralogues, fixed in this genome after the salmonid-specific 4th whole genome duplication, may have a role in the setting up of the glucose-intolerant phenotype in this carnivorous species. This should be due to the sub- or neo-functionalization of their regulation. In the present short communication we thus addressed the question of the regulation of these genes by insulin, hormone involved in the glucose homeostasis, and its interaction with glucose and amino acids in vitro. The stimulation of trout hepatocytes with insulin revealed an atypical up-regulation of g6pcb2 ohnologues and confirmed the sub- or neo-functionalization of the five g6pc genes at least at the regulatory level. Intriguingly, when hepatocytes were cultured with high levels of glucose and/or AAs in presence of insulin, most of the g6pc paralogues were up-regulated. It strongly suggested a cross-talk between insulin and nutrients for the regulation of these genes. Moreover these results strengthened the idea that g6pc duplicated genes may significantly contribute to the setting up of the glucose-intolerant phenotype in trout via their atypical regulation by insulin alone or in interaction with nutrients. These findings open new perspectives to better understand in vivo glucose-intolerant phenotype in trout fed a high carbohydrate diet. PMID:26896939

  16. Hepatocyte growth factor (HGF) signals through SHP2 to regulate primary mouse myoblast proliferation

    SciTech Connect

    Li, Ju; Reed, Sarah A.; Johnson, Sally E.

    2009-08-01

    Niche localized HGF plays an integral role in G{sub 0} exit and the return to mitotic activity of adult skeletal muscle satellite cells. HGF actions are regulated by MET initiated intracellular signaling events that include recruitment of SHP2, a protein tyrosine phosphatase. The importance of SHP2 in HGF-mediated signaling was examined in myoblasts and primary cultures of satellite cells. Myoblasts stably expressing SHP2 (23A2-SHP2) demonstrate increased proliferation rates by comparison to controls or myoblasts expressing a phosphatase-deficient SHP2 (23A2-SHP2DN). By comparison to 23A2 myoblasts, treatment of 23A2-SHP2 cells with HGF does not further increase proliferation rates and 23A2-SHP2DN myoblasts are unresponsive to HGF. Importantly, the effects of SHP2 are independent of downstream ERK1/2 activity as inclusion of PD98059 does not blunt the HGF-induced proliferative response. SHP2 function was further evaluated in primary satellite cell cultures. Ectopic expression of SHP2 in satellite cells tends to decrease proliferation rates and siSHP2 causes an increase the percentage of dividing myogenic cells. Interestingly, treatment of satellite cells with high concentrations of HGF (50 ng/ml) inhibits proliferation, which can be overcome by knockdown of SHP2. From these results, we conclude that HGF signals through SHP2 in myoblasts and satellite cells to directly alter proliferation rates.

  17. Angiopoietin-like 3 regulates hepatocyte proliferation and lipid metabolism in zebrafish

    SciTech Connect

    Lee, So-Hyun; So, Ju-Hoon; Kim, Hyun-Taek; Choi, Jung-Hwa; Lee, Mi-Sun; Choi, Seok-Yong; Kim, Cheol-Hee; Kim, Min Jung

    2014-04-18

    Highlights: • angptl3 is specifically expressed in the liver of developing zebrafish. • Knockdown of Angptl3 decreases liver size in developing zebrafish. • Knockdown of zebrafish Angptl3 elicits a hypocholesterolemia phenotype. - Abstract: Loss-of-function mutations in angiopoietin-like 3 (ANGPTL3) cause familial hypobetalipoproteinemia type 2 (FHBL2) in humans. ANGPTL3 belongs to the angiopoietin-like family, the vascular endothelial growth factor family that is structurally similar to angiopoietins and is known for a regulator of lipid and glucose metabolism, although it is unclear how mutations in ANGPTL3 lead to defect in liver development in the vertebrates. We report here that angptl3 is primarily expressed in the zebrafish developing liver and that morpholino (MO) knockdown of Angptl3 reduces the size of the developing liver, which is caused by suppression of cell proliferation, but not by enhancement of apoptosis. However, MO knockdown of Angptl3 did not alter angiogenesis in the developing liver. Additionally, disruption of zebrafish Angptl3 elicits the hypocholesterolemia phenotype that is characteristic of FHBL2 in humans. Together, our findings propose a novel role for Angptl3 in liver cell proliferation and maintenance during zebrafish embryogenesis. Finally, angptl3 morphants will serve as a good model for understanding the pathophysiology of FHBL2.

  18. DHA down-regulates phenobarbital-induced cytochrome P450 2B1 gene expression in rat primary hepatocytes by attenuating CAR translocation

    SciTech Connect

    Li, C.-C.; Lii, C.-K.; Liu, K.-L.; Yang, J.-J.; Chen, H.-W.

    2007-12-15

    The constitutive androstane receptor (CAR) plays an important role in regulating the expression of detoxifying enzymes, including cytochrome P450 2B (CYP 2B). Phenobarbital (PB) induction of human CYP 2B6 and mouse CYP 2b10 has been shown to be mediated by CAR. Our previous study showed that PB-induced CYP 2B1 expression in rat primary hepatocytes is down-regulated by both n-6 and n-3 polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid (DHA); however, the mechanism for this down-regulation by DHA was previously unknown. The objective of the present study was to determine whether change in CAR translocation is involved in the down-regulation by n-6 and n-3 PUFAs of PB-induced CYP 2B1 expression in rat primary hepatocytes. We used 100 {mu}M arachidonic acid, linoleic acid, eicosapentaenoic acid, and DHA to test this hypothesis. PB triggered the translocation of CAR from the cytosol into the nucleus in a dose-dependent and time-dependent manner in our hepatocyte system, and the CAR distribution in rat primary hepatocytes was significantly affected by DHA. DHA treatment decreased PB-inducible accumulation of CAR in the nuclear fraction and increased it in the cytosolic fraction in a dose-dependent manner. The down-regulation of CYP 2B1 expression by DHA occurred in a dose-dependent manner, and a similar pattern was found for the nuclear accumulation of CAR. The results of immunoprecipitation showed a CAR/RXR heterodimer bound to nuclear receptor binding site 1 (NR-1) of the PB-responsive enhancer module (PBREM) of the CYP 2B1gene. The EMSA results showed that PB-induced CAR binding to NR-1 was attenuated by DHA. Taken together, these results suggest that attenuation of CAR translocation and decreased subsequent binding to NR-1 are involved in DHA's down-regulation of PB-induced CYP 2B1 expression.

  19. Interaction of hepatocyte nuclear factors in transcriptional regulation of tissue specific hormonal expression of human multidrug resistance-associated protein 2 (abcc2)

    SciTech Connect

    Qadri, Ishtiaq Hu, L.-J.; Iwahashi, Mieko; Al-Zuabi, Subhi; Quattrochi, Linda C.; Simon, Francis R.

    2009-02-01

    Multidrug resistance-associated protein 2 (MRP2) (ABCC2) is an ATP-binding cassette membrane protein located primarily on apical surface of hepatocytes that mediates transport of conjugated xenobiotics and endogenous compounds into bile. MRP2 is highly expressed in hepatocytes, and at lower levels in small intestines, stomach and kidney. Previous reports have characterized mammalian MRP2 promoters, but none have established the molecular mechanism(s) involved in liver enriched expression. This study aims to investigate the mechanism of hepatic MRP2 regulation. A 2130 bp of MRP2 promoter was cloned from PAC-1 clone P108G1-7, to identify putative liver specific/hormone responsive functional DNA binding sites. Using deletion analysis, site specific mutagenesis and co-transfection studies, liver specific expression was determined. MRP2 promoter-LUC constructs were highly expressed in liver cell lines compared to non-liver cells. The region extending from - 3 to+ 458 bp of MRP2 promoter starting from AUG contained the potential binding sites for CAAATT box enhancer binding protein (C/EBP), hepatocytes nuclear factor 1, 3 and 4 (HNF1, HNF3, and HNF4. Only HNF1 and HNF4 co-transfection with MRP2 luciferase increased expression. Site specific mutational analysis of HNF1 binding site indicated an important role for HNF1{alpha}. HNF4{alpha} induction of MRP2 was independent of HNF1 binding site. C/EBP, HNF3, and HNF6 inhibited HNF1{alpha} while HNF4{alpha} induced MRP2 luciferase expression and glucocorticoids stimulated MRP2 expression. This study emphasizes the complex regulation of MRP2 with HNF1{alpha} and HNF4{alpha} playing a central role. The coordinated regulation of xenobiotic transporters and oxidative conjugation may determine the adaptive responses to cellular detoxification processes.

  20. Multiple hepatocyte-enriched nuclear factors function in the regulation of transthyretin and. alpha. 1-antitrypsin genes

    SciTech Connect

    Costa, R.H. ); Grayson, D.R. ); Darnell, J.E. Jr. )

    1989-04-01

    Transthyretin (TTR) and {alpha}1-antitrypsin ({alpha}1-AT) are expressed at high levels in the liver and also in at least one other cell type. The authors report here a detailed analysis of the proximal regulatory region of the TTR gene, which has uncovered two new DNA-binding factors that are present mainly (or only) in hepatocytes. One of these new factors, hepatocyte nuclear factor 3 (HNF-3), binds to two sites that are crucial in TTR expression as well as to two additional sites in the {alpha}1-AT proximal enhancer region. The second new factor, HNF-4, binds to two sites in TTR that are required for gene activity. The authors had previously identified binding sites for another hepatocyte-enriched DNA-binding protein (C/EBP or a relative thereof), and additional promoter-proximal sites for that protein in both TTR and {alpha}1-AT are also reported here. From these results it seems clear that cell-specific expression is not simply the result of a single cell-specific factor for each gene but the results of a combination of such factors. The variation and distribution of such factors among different cell types could be an important basis for the coordinate expression of the TTR and {alpha}1-AT genes in the liver or the discordant transcriptional activation of these genes in a few other cell types. The identification of such cell-enriched factors is a necessary prelude to understanding the basis for cell specificity.

  1. Carboxylesterase 1 Is Regulated by Hepatocyte Nuclear Factor 4α and Protects Against Alcohol- and MCD diet-induced Liver Injury

    PubMed Central

    Xu, Jiesi; Xu, Yang; Li, Yuanyuan; Jadhav, Kavita; You, Min; Yin, Liya; Zhang, Yanqiao

    2016-01-01

    The liver is a major organ that controls hepatic and systemic homeostasis. Dysregulation of liver metabolism may cause liver injury. Previous studies have demonstrated that carboxylesterase 1 (CES1) regulates hepatic triglyceride metabolism and protects against liver steatosis. In the present study, we investigated whether CES1 played a role in the development of alcoholic liver disease (ALD) and methionine and choline-deficient (MCD) diet-induced liver injury. Both hepatocyte nuclear factor 4α (HNF4α) and CES1 were markedly reduced in patients with alcoholic steatohepatitis. Alcohol repressed both HNF4α and CES1 expression in primary hepatocytes. HNF4α regulated CES1 expression by directly binding to the proximal promoter of CES1. Global inactivation of CES1 aggravated alcohol- or MCD diet-induced liver inflammation and liver injury, likely as a result of increased production of acetaldehyde and reactive oxygen species and mitochondrial dysfunctions. Knockdown of hepatic CES1 exacerbated ethanol-induced steatohepatitis. These data indicate that CES1 plays a crucial role in protection against alcohol- or MCD diet-induced liver injury. PMID:27075303

  2. Carboxylesterase 1 Is Regulated by Hepatocyte Nuclear Factor 4α and Protects Against Alcohol- and MCD diet-induced Liver Injury.

    PubMed

    Xu, Jiesi; Xu, Yang; Li, Yuanyuan; Jadhav, Kavita; You, Min; Yin, Liya; Zhang, Yanqiao

    2016-01-01

    The liver is a major organ that controls hepatic and systemic homeostasis. Dysregulation of liver metabolism may cause liver injury. Previous studies have demonstrated that carboxylesterase 1 (CES1) regulates hepatic triglyceride metabolism and protects against liver steatosis. In the present study, we investigated whether CES1 played a role in the development of alcoholic liver disease (ALD) and methionine and choline-deficient (MCD) diet-induced liver injury. Both hepatocyte nuclear factor 4α (HNF4α) and CES1 were markedly reduced in patients with alcoholic steatohepatitis. Alcohol repressed both HNF4α and CES1 expression in primary hepatocytes. HNF4α regulated CES1 expression by directly binding to the proximal promoter of CES1. Global inactivation of CES1 aggravated alcohol- or MCD diet-induced liver inflammation and liver injury, likely as a result of increased production of acetaldehyde and reactive oxygen species and mitochondrial dysfunctions. Knockdown of hepatic CES1 exacerbated ethanol-induced steatohepatitis. These data indicate that CES1 plays a crucial role in protection against alcohol- or MCD diet-induced liver injury. PMID:27075303

  3. Functional analysis and transcriptional regulation of porcine six transmembrane epithelial antigen of prostate 4 (STEAP4) gene and its novel variant in hepatocytes.

    PubMed

    Wang, S B; Lei, T; Zhou, L L; Zheng, H L; Zeng, C P; Liu, N; Yang, Z Q; Chen, X D

    2013-03-01

    Six-transmembrane epithelial antigen of prostate 4 (STEAP4) plays a critical role in modulating inflammatory response and protecting metabolic function. However, the role of STEAP4 in hepatocytes is not well understood, and the mechanism of STEAP4 action remains elusive. Here, we report the molecular characterization of porcine STEAP4 and its novel splice variant (STEAP4v), then the metabolic and anti-inflammatory roles of porcine STEAP4 and STEAP4v were investigated in HepG2 liver cells. The results revealed that overexpression of STEAP4, but not STEAP4v, suppresses triglyceride (TG) content and ameliorates the up-regulation of the transcription of the genes necessary for de novo lipogenesis and gluconeogenesis elicited by FFAs treatment. In RAW264.7 macrophage cells, transient transfection of STEAP4v, to a greater extent than STEAP4, repressed the transcription of TNFα and IL-6. In HepG2 cells with LPS treatment, the endogenous mRNA and protein levels of STEAP4 and STEAP4v were up-regulated, which was accompanied by a concurrent increase in C/EBPβ mRNA and protein levels. Thirdly, the functional regulation of STEAP4 was explored, which revealed that the porcine STEAP4 promoter activity was significantly up-regulated by C/EBPβ. The progressive deletions and mutations demonstrated that the C/EBPβ binding motif situated at -73/-59 bp is an essential component required for promoter activity of STEAP4 gene. Chromatin immunoprecipitation (ChIP) assays determined that C/EBPβ can directly interact with the steap4 promoter DNA. In conclusion, our data demonstrated that C/EBPβ directly regulates the roles of STEAP4 and its novel variant in attenuating lipogenesis, gluconeogenesis or/and inflammation elicited by FFAs or LPS in hepatocytes. PMID:23262293

  4. Strategies for immortalization of primary hepatocytes

    PubMed Central

    Eva, Ramboer; Bram, De Craene; Joery, De Kock; Tamara, Vanhaecke; Geert, Berx; Vera, Rogiers; Mathieu, Vinken

    2014-01-01

    The liver has the unique capacity to regenerate in response to a damaging event. Liver regeneration is hereby largely driven by hepatocyte proliferation, which in turn relies on cell cycling. The hepatocyte cell cycle is a complex process that is tightly regulated by several well-established mechanisms. In vitro, isolated hepatocytes do not longer retain this proliferative capacity. However, in vitro cell growth can be boosted by immortalization of hepatocytes. Well-defined immortalization genes can be artificially overexpressed in hepatocytes or the cells can be conditionally immortalized leading to controlled cell proliferation. This paper discusses the current immortalization techniques and provides a state-of-the-art overview of the actually available immortalized hepatocyte-derived cell lines and their applications. PMID:24911463

  5. Gene-specific alterations of hepatic nuclear receptor regulated gene expression by ligand activation or hepatocyte-selective knockout inhibition of RXRα signaling during inflammation

    PubMed Central

    Kosters, Astrid; Tian, Feng; Wan, Yvonne Yu-Jie; Karpen, Saul J.

    2013-01-01

    Background Inflammation leads to transcriptional downregulation of many hepatic genes, particulary those activated by RXRα-heterodimers. Inflammation-mediated reduction of nuclear RXRα levels is a main factor in reduced nuclear receptor (NR)–regulated hepatic gene expression, eventually leading to cholestasis and liver damage. Aim To investigate roles for RXRα in hepatic gene expression during inflammation, using two complementary mouse models: ligand–activation of RXRα, and in mice expressing hepatocyte-specific expression of RXRα missing its DNA-binding-domain (DBD; hs-RxrαΔex4−/−) Methods To activate RXRα, mice were gavage-fed with LG268 or vehicle for 5 days. To inhibit RXRα function, hs-RxrαΔex4−/− were used. All mice were IP-injected with LPS or saline for 16 hrs prior to analysis of hepatic RNA, protein and NR-DNA binding. Results LG268-treatment attenuated the LPS-mediated reductions of several RXRα-regulated genes, coinciding with maintained RXRα occupancy in both Bsep and Ostβ promoters. Lacking full hepatocyte-RXRα function (hs-RxrαΔex4−/− mice) led to enhancement of LPS-mediated changes in gene expression, but surprisingly, maintenance of RNA levels of some RXRα-regulated genes. Investigations revealed that Hs-Rxrα−/− hepatocytes expressed an internally-truncated, ~44 kDa, RXRα-form. DNA-binding capacity of NR-heterodimers was equivalent in wt and hs-RxrαΔex4−/− livers, but reduced by LPS in both. ChIP-QPCR revealed reduced RXRα occupancy to the Bsep RXRα:FXR site was reduced, but not absent, in hs-RxrαΔex4−/− livers. Conclusions There are differential regulatory roles for hepatic RXRα, both in basal and inflammatory states, suggesting new and complex multi-domain roles for RXRα in regulating hepatic gene expression. Moreover, there is an unexpected non-obligate role for the DBD of RXRα. PMID:22098603

  6. Nitric Oxide and Redox Regulation in the Liver: Part II Redox biology in Pathologic Hepatocytes and Implications for intervention

    PubMed Central

    Diesen, Diana L.; Kuo, Paul C.

    2009-01-01

    Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are created in normal hepatocytes and are critical for normal physiological processes including oxidative respiration, growth, regeneration, apoptosis, and microsomal defense. When the levels of oxidation products exceed the capacity of normal antioxidant systems, oxidative stress occurs. This type of stress, in the form of ROS and RNS, can be damaging to all liver cells, including hepatocytes, Kupffer cells, stellate cells, and endothelial cells, through induction of inflammation, ischemia, fibrosis, necrosis, apoptosis, or through malignant transformation by damaging lipids, proteins, and/or DNA. In part I of this review, we will discuss basic redox biology in the liver, including a review of ROS, RNS, and antioxidants, with a focus on nitric oxide as a common source of RNS. We will then review the evidence for oxidative stress as a mechanism of liver injury in hepatitis (alcoholic, viral, non-alcoholic). In part II of this review, we will review oxidative stress in common pathophysiological conditions including ischemia/reperfusion injury, fibrosis, hepatocellular carcinoma, iron overload, Wilson’s disease, sepsis and acetaminophen overdose. Finally, biomarkers, proteomic, and antioxidant therapies will be discussed as areas for future therapeutic interventions. PMID:20400112

  7. Mechanism of a transcriptional cross talk between transforming growth factor-beta-regulated Smad3 and Smad4 proteins and orphan nuclear receptor hepatocyte nuclear factor-4.

    PubMed

    Chou, Wan-Chih; Prokova, Vassiliki; Shiraishi, Keiko; Valcourt, Ulrich; Moustakas, Aristidis; Hadzopoulou-Cladaras, Margarita; Zannis, Vassilis I; Kardassis, Dimitris

    2003-03-01

    We have shown previously that the transforming growth factor-beta (TGFbeta)-regulated Sma-Mad (Smad) protein 3 and Smad4 proteins transactivate the apolipoprotein C-III promoter in hepatic cells via a hormone response element that binds the nuclear receptor hepatocyte nuclear factor 4 (HNF-4). In the present study, we show that Smad3 and Smad4 but not Smad2 physically interact with HNF-4 via their Mad homology 1 domains both in vitro and in vivo. The synergistic transactivation of target promoters by Smads and HNF-4 was shown to depend on the specific promoter context and did not require an intact beta-hairpin/DNA binding domain of the Smads. Using glutathione S-transferase interaction assays, we established that two regions of HNF-4, the N-terminal activation function 1 (AF-1) domain (aa 1-24) and the C-terminal F domain (aa 388-455) can mediate physical Smad3/HNF-4 interactions in vitro. In vivo, Smad3 and Smad4 proteins enhanced the transactivation function of various GAL4-HNF-4 fusion proteins via the AF-1 and the adjacent DNA binding domain, whereas a single tyrosine to alanine substitution in AF-1 abolished coactivation by Smads. The findings suggest that the transcriptional cross talk between the TGFbeta-regulated Smads and HNF-4 is mediated by specific functional domains in the two types of transcription factors. Furthermore, the specificity of this interaction for certain target promoters may play an important role in various hepatocyte functions, which are regulated by TGFbeta and the Smads. PMID:12631740

  8. Differential Regulation of Gene Expression by Cholesterol Biosynthesis Inhibitors That Reduce (Pravastatin) or Enhance (Squalestatin 1) Nonsterol Isoprenoid Levels in Primary Cultured Mouse and Rat Hepatocytes.

    PubMed

    Rondini, Elizabeth A; Duniec-Dmuchowski, Zofia; Cukovic, Daniela; Dombkowski, Alan A; Kocarek, Thomas A

    2016-08-01

    Squalene synthase inhibitors (SSIs), such as squalestatin 1 (SQ1), reduce cholesterol biosynthesis but cause the accumulation of isoprenoids derived from farnesyl pyrophosphate (FPP), which can modulate the activity of nuclear receptors, including the constitutive androstane receptor (CAR), farnesoid X receptor, and peroxisome proliferator-activated receptors (PPARs). In comparison, 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (e.g., pravastatin) inhibit production of both cholesterol and nonsterol isoprenoids. To characterize the effects of isoprenoids on hepatocellular physiology, microarrays were used to compare orthologous gene expression from primary cultured mouse and rat hepatocytes that were treated with either SQ1 or pravastatin. Compared with controls, 47 orthologs were affected by both inhibitors, 90 were affected only by SQ1, and 51 were unique to pravastatin treatment (P < 0.05, ≥1.5-fold change). When the effects of SQ1 and pravastatin were compared directly, 162 orthologs were found to be differentially coregulated between the two treatments. Genes involved in cholesterol and unsaturated fatty acid biosynthesis were up-regulated by both inhibitors, consistent with cholesterol depletion; however, the extent of induction was greater in rat than in mouse hepatocytes. SQ1 induced several orthologs associated with microsomal, peroxisomal, and mitochondrial fatty acid oxidation and repressed orthologs involved in cell cycle regulation. By comparison, pravastatin repressed the expression of orthologs involved in retinol and xenobiotic metabolism. Several of the metabolic genes altered by isoprenoids were inducible by a PPARα agonist, whereas cytochrome P450 isoform 2B was inducible by activators of CAR. Our findings indicate that SSIs uniquely influence cellular lipid metabolism and cell cycle regulation, probably due to FPP catabolism through the farnesol pathway. PMID:27225895

  9. Low-dose irradiation promotes Rad51 expression by down-regulating miR-193b-3p in hepatocytes.

    PubMed

    Lee, Eon-Seok; Won, Yeo Jin; Kim, Byoung-Chul; Park, Daeui; Bae, Jin-Han; Park, Seong-Joon; Noh, Sung Jin; Kang, Yeong-Rok; Choi, Si Ho; Yoon, Je-Hyun; Heo, Kyu; Yang, Kwangmo; Son, Tae Gen

    2016-01-01

    Current evidence indicates that there is a relationship between microRNA (miRNA)-mediated gene silencing and low-dose irradiation (LDIR) responses. Here, alterations of miRNA expression in response to LDIR exposure in male BALB/c mice and three different types of hepatocytes were investigated. The miRNome of the LDIR-exposed mouse spleens (0.01 Gy, 6.5 mGy/h) was analyzed, and the expression of miRNA and mRNA was validated by qRT-PCR. Western blotting, chromatin immunoprecipitation (ChIP), and luciferase assays were also performed to evaluate the interaction between miRNAs and their target genes and to gain insight into the regulation of miRNA expression. The expression of miRNA-193b-3p was down-regulated in the mouse spleen and liver and in various hepatocytes (NCTC, Hepa, and HepG2 cell lines) in response to LDIR. The down-regulation of miR-193b-3p expression was caused by histone deacetylation on the miR-193b-3p promoter in the HepG2 cells irradiated with 0.01 Gy. However, the alteration of histone deacetylation and miR-193b-3p and Rad51 expression in response to LDIR was restored by pretreatment with N-acetyl-cyctein. In conclusion, we provide evidence that miRNA responses to LDIR include the modulation of cellular stress responses and repair mechanisms. PMID:27225532

  10. Low-dose irradiation promotes Rad51 expression by down-regulating miR-193b-3p in hepatocytes

    PubMed Central

    Lee, Eon-Seok; Won, Yeo Jin; Kim, Byoung-Chul; Park, Daeui; Bae, Jin-Han; Park, Seong-Joon; Noh, Sung Jin; Kang, Yeong-Rok; Choi, Si Ho; Yoon, Je-Hyun; Heo, Kyu; Yang, Kwangmo; Son, Tae Gen

    2016-01-01

    Current evidence indicates that there is a relationship between microRNA (miRNA)-mediated gene silencing and low-dose irradiation (LDIR) responses. Here, alterations of miRNA expression in response to LDIR exposure in male BALB/c mice and three different types of hepatocytes were investigated. The miRNome of the LDIR-exposed mouse spleens (0.01 Gy, 6.5 mGy/h) was analyzed, and the expression of miRNA and mRNA was validated by qRT-PCR. Western blotting, chromatin immunoprecipitation (ChIP), and luciferase assays were also performed to evaluate the interaction between miRNAs and their target genes and to gain insight into the regulation of miRNA expression. The expression of miRNA-193b-3p was down-regulated in the mouse spleen and liver and in various hepatocytes (NCTC, Hepa, and HepG2 cell lines) in response to LDIR. The down-regulation of miR-193b-3p expression was caused by histone deacetylation on the miR-193b-3p promoter in the HepG2 cells irradiated with 0.01 Gy. However, the alteration of histone deacetylation and miR-193b-3p and Rad51 expression in response to LDIR was restored by pretreatment with N-acetyl-cyctein. In conclusion, we provide evidence that miRNA responses to LDIR include the modulation of cellular stress responses and repair mechanisms. PMID:27225532

  11. Low-dose irradiation promotes Rad51 expression by down-regulating miR-193b-3p in hepatocytes

    NASA Astrophysics Data System (ADS)

    Lee, Eon-Seok; Won, Yeo Jin; Kim, Byoung-Chul; Park, Daeui; Bae, Jin-Han; Park, Seong-Joon; Noh, Sung Jin; Kang, Yeong-Rok; Choi, Si Ho; Yoon, Je-Hyun; Heo, Kyu; Yang, Kwangmo; Son, Tae Gen

    2016-05-01

    Current evidence indicates that there is a relationship between microRNA (miRNA)-mediated gene silencing and low-dose irradiation (LDIR) responses. Here, alterations of miRNA expression in response to LDIR exposure in male BALB/c mice and three different types of hepatocytes were investigated. The miRNome of the LDIR-exposed mouse spleens (0.01 Gy, 6.5 mGy/h) was analyzed, and the expression of miRNA and mRNA was validated by qRT-PCR. Western blotting, chromatin immunoprecipitation (ChIP), and luciferase assays were also performed to evaluate the interaction between miRNAs and their target genes and to gain insight into the regulation of miRNA expression. The expression of miRNA-193b-3p was down-regulated in the mouse spleen and liver and in various hepatocytes (NCTC, Hepa, and HepG2 cell lines) in response to LDIR. The down-regulation of miR-193b-3p expression was caused by histone deacetylation on the miR-193b-3p promoter in the HepG2 cells irradiated with 0.01 Gy. However, the alteration of histone deacetylation and miR-193b-3p and Rad51 expression in response to LDIR was restored by pretreatment with N-acetyl-cyctein. In conclusion, we provide evidence that miRNA responses to LDIR include the modulation of cellular stress responses and repair mechanisms.

  12. Pancreatic B-13 Cell Trans-Differentiation to Hepatocytes Is Dependent on Epigenetic-Regulated Changes in Gene Expression

    PubMed Central

    Fairhall, Emma A.; Charles, Michelle A.; Probert, Philip M. E.; Wallace, Karen; Gibb, Jennifer; Ravindan, Chandni; Soloman, Martin; Wright, Matthew C.

    2016-01-01

    The proliferative B-13 pancreatic cell line is unique in its ability to generate functional hepatocyte-like (B-13/H) cells in response to exposure to glucocorticoid. In these studies, quantitatively comparable hepatic levels of liver-specific and liver-enriched transcription factor and hepatocyte defining mRNA transcripts were expressed after 10–14 days continuous treatment with glucocorticoid. This conversion in phenotype was associated with increased Gr-α mRNA expression and translation of a functional N-terminally truncated variant protein that localized to the nucleus in B-13/H cells. A short (6 hours) pulse exposure to glucocorticoid was also sufficient to transiently activate the Gr and irreversibly drive near identical conversion to B-13/H cells. Examination of epigenetic-related mechanisms demonstrated that B-13 DNA was rapidly methylated and de-methylated over the initial 2 days in response to both continuous or pulse exposure with glucocorticoid. DNA methylation and glucocorticoid-dependent conversion to an hepatic B-13/H phenotype was blocked by the methylation inhibitor, 5-azacytidine. Conversion to an hepatic B-13/H phenotype was also blocked by histone deacetylase inhibitors. Previous experiments have identified N-terminal Sgk1 variant proteins as pivotal to the mechanism(s) associated with pancreatic–hepatic differentiation. Both continuous and pulse exposure to DEX was sufficient to result in a near-similar robust transcriptional increase in Sgk1c mRNA expression from undetectable levels in B-13 cells. Notably, expression of Sgk1c mRNA remained constitutive 14 days later; including after pulse exposure to glucocorticoid and this induction was inhibited by 5-azacytidine or by histone deacetylase inhibitors. These data therefore suggest that exposing B-13 cells to glucocorticoid results in a Gr-dependent pulse in DNA methylation and likely other epigenetic changes such as histone modifications that leads to constitutive expression of Sgk1c and

  13. Rifampin Regulation of Drug Transporters Gene Expression and the Association of MicroRNAs in Human Hepatocytes

    PubMed Central

    Benson, Eric A.; Eadon, Michael T.; Desta, Zeruesenay; Liu, Yunlong; Lin, Hai; Burgess, Kimberly S.; Segar, Matthew W.; Gaedigk, Andrea; Skaar, Todd C.

    2016-01-01

    Membrane drug transporters contribute to the disposition of many drugs. In human liver, drug transport is controlled by two main superfamilies of transporters, the solute carrier transporters (SLC) and the ATP Binding Cassette transporters (ABC). Altered expression of these transporters due to drug-drug interactions can contribute to differences in drug exposure and possibly effect. In this study, we determined the effect of rifampin on gene expression of hundreds of membrane transporters along with all clinically relevant drug transporters. Methods: In this study, primary human hepatocytes (n = 7 donors) were cultured and treated for 24 h with rifampin and vehicle control. RNA was isolated from the hepatocytes, mRNA expression was measured by RNA-seq, and miRNA expression was analyzed by Taqman OpenArray. The effect of rifampin on the expression of selected transporters was also tested in kidney cell lines. The impact of rifampin on the expression of 410 transporter genes from 19 different transporter gene families was compared with vehicle control. Results: Expression patterns of 12 clinically relevant drug transporter genes were changed by rifampin (FDR < 0.05). For example, the expressions of ABCC2, ABCB1, and ABCC3 were increased 1.9-, 1.7-, and 1.2-fold, respectively. The effects of rifampin on four uptake drug transporters (SLCO1B3, SLC47A1, SLC29A1, SLC22A9) were negatively correlated with the rifampin effects on specific microRNA expression (SLCO1B3/miR-92a, SLC47A1/miR-95, SLC29A1/miR-30d#, and SLC22A9/miR-20; r < −0.79; p < 0.05). Seven hepatic drug transporter genes (SLC22A1, SLC22A5, SLC15A1, SLC29A1, SLCO4C1, ABCC2, and ABCC4), whose expression was altered by rifampin in hepatocytes, were also present in a renal proximal tubular cell line, but in renal cells rifampin did not alter their gene expression. PXR expression was very low in the kidney cells; this may explain why rifampin induces gene expression in a tissue-specific manner. Conclusion

  14. Hepatocyte-specific deletion of hepatocyte nuclear factor-4α in adult mice results in increased hepatocyte proliferation

    PubMed Central

    Walesky, Chad; Gunewardena, Sumedha; Terwilliger, Ernest F.; Edwards, Genea; Borude, Prachi

    2013-01-01

    Hepatocyte nuclear factor-4α (HNF4α) is known as the master regulator of hepatocyte differentiation. Recent studies indicate that HNF4α may inhibit hepatocyte proliferation via mechanisms that have yet to be identified. Using a HNF4α knockdown mouse model based on delivery of inducible Cre recombinase via an adeno-associated virus 8 viral vector, we investigated the role of HNF4α in the regulation of hepatocyte proliferation. Hepatocyte-specific deletion of HNF4α resulted in increased hepatocyte proliferation. Global gene expression analysis showed that a majority of the downregulated genes were previously known HNF4α target genes involved in hepatic differentiation. Interestingly, ≥500 upregulated genes were associated with cell proliferation and cancer. Furthermore, we identified potential negative target genes of HNF4α, many of which are involved in the stimulation of proliferation. Using chromatin immunoprecipitation analysis, we confirmed binding of HNF4α at three of these genes. Furthermore, overexpression of HNF4α in mouse hepatocellular carcinoma cells resulted in a decrease in promitogenic gene expression and cell cycle arrest. Taken together, these data indicate that, apart from its role in hepatocyte differentiation, HNF4α actively inhibits hepatocyte proliferation by repression of specific promitogenic genes. PMID:23104559

  15. Hepatocyte growth factor-induced up-regulation of Twist drives epithelial-mesenchymal transition in a canine mammary tumour cell line.

    PubMed

    Yoshida, Kota; Choisunirachon, Nan; Saito, Tomochika; Matsumoto, Kaori; Saeki, Kohei; Mochizuki, Manabu; Nishimura, Ryohei; Sasaki, Nobuo; Nakagawa, Takayuki

    2014-12-01

    Epithelial-mesenchymal transition (EMT) is a crucial step in tumour progression. However, the molecular mechanisms underlying EMT in canine tumours remain to be elucidated. In this study, the similarity or difference in the molecular mechanism of EMT in canine cells was evaluated and compared with that reported in human and mouse cells. We used eight cell lines derived from canine mammary cancers. Stimulation with hepatocyte growth factor (HGF) increased cell motility and changed EMT-related markers towards mesenchyme in CHMm cell line. These changes were accompanied by an increase in Twist expression and did not occur in CHMm transfected with Twist siRNA, indicating that Twist plays a key role in this phenomenon in CHMm. However, the down-regulation of E-cadherin was not observed by HGF stimulation. Further studies are required to elucidate the difference between human and canine Twist. PMID:25278141

  16. Monoacylglycerol O-acyltransferase 1 is regulated by peroxisome proliferator-activated receptor γ in human hepatocytes and increases lipid accumulation

    SciTech Connect

    Yu, Jung Hwan; Lee, Yoo Jeong; Kim, Hyo Jung; Choi, Hyeonjin; Choi, Yoonjeong; Seok, Jo Woon; Kim, Jae-woo

    2015-05-08

    Monoacylglycerol O-acyltransferase (MGAT) is an enzyme that is involved in triglyceride synthesis by catalyzing the formation of diacylglycerol from monoacylglycerol and fatty acyl CoAs. Recently, we reported that MGAT1 has a critical role in hepatic TG accumulation and that its suppression ameliorates hepatic steatosis in a mouse model. However, the function of MGAT enzymes in hepatic lipid accumulation has not been investigated in humans. Unlike in rodents, MGAT3 as well as MGAT1 and MGAT2 are present in humans. In this study, we evaluated the differences between MGAT subtypes and their association with peroxisome proliferator-activated receptor γ (PPARγ), a regulator of mouse MGAT1 expression. In human primary hepatocytes, basal expression of MGAT1 was lower than that of MGAT2 or MGAT3, but was strongly induced by PPARγ overexpression. A luciferase assay as well as an electromobility shift assay revealed that human MGAT1 promoter activity is driven by PPARγ by direct binding to at least two regions of the promoter in 293T and HepG2 cells. Moreover, siRNA-mediated suppression of MGAT1 expression significantly attenuated lipid accumulation by PPARγ overexpression in HepG2 cells, as evidenced by oil-red-O staining. These results suggest that human MGAT1 has an important role in fatty liver formation as a target gene of PPARγ, and blocking MGAT1 activity could be an efficient therapeutic way to reduce nonalcoholic fatty liver diseases in humans. - Highlights: • PPARγ promotes MGAT1 expression in human primary hepatocytes. • PPARγ directly regulates MGAT1 promoter activity. • Human MGAT1 promoter has at least two PPARγ-binding elements. • Inhibition of MGAT1 expression attenuates hepatic lipid accumulation in humans.

  17. 1. GENERAL VIEW. RED BARN WITH HEX SIGNS. DATE AND ...

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

    1. GENERAL VIEW. RED BARN WITH HEX SIGNS. DATE AND NAME PAINTED IN CENTER OF FRONT WALL. SIGNS ARE LIGHT GREEN AND YELLOW IN WHITE CIRCLES. LOWER ONES WERE PAINED OVER OLD TEAR DROP SWASTIKA. DOORS AND WINDOWS HAVE PEDIMENT HEADS - H. & S. Hoffman Barn (1853), Pottstown, Montgomery County, PA

  18. Hawthorn (Crataegus oxyacantha L.) bark extract regulates antioxidant response element (ARE)-mediated enzyme expression via Nrf2 pathway activation in normal hepatocyte cell line.

    PubMed

    Krajka-Kuźniak, Violetta; Paluszczak, Jarosław; Oszmiański, Jan; Baer-Dubowska, Wanda

    2014-04-01

    Hawthorn (Crataegus oxyacantha L.), a plant used in traditional medicine, is a rich source of procyanidins which have been reported to exhibit antioxidant and anti-carcinogenic activity. In this study, we assessed the effect of hawthorn bark extract (HBE) on Nrf2 pathway activation in THLE-2 and HepG2 cells. Treatment with 1.1 µg/mL, 5.5 µg/mL and 11 µg/mL of HBE resulted in the translocation of Nrf2 from the cytosol to the nucleus in both cell lines; however, the accumulation of phosphorylated Nrf2 was observed only in THLE-2. Accordingly, treatment of cells with HBE was associated with an increase in the mRNA and protein level of such Nrf2-dependent genes as glutathione S-transferases (GSTA, GSTP, GSTM, GSTT), NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) (0.2-1.1-fold change, p < 0.05), however, only in normal THLE-2 hepatocytes. The induction of NQO1 correlated with an increased level of p53 (0.21-0.42-fold change, p < 0.05). These effects may be related to induction of phosphorylation of upstream ERK and JNK kinases. Collectively, the results suggest that the Nrf2/ARE pathway may play an important role in the regulation of procyanidin-mediated antioxidant/detoxifying effects in hepatocytes, and this may explain the hepatoprotective and chemopreventive properties of these phytochemicals. PMID:23843400

  19. Epigenetically regulated miR-449a enhances hepatitis B virus replication by targeting cAMP-responsive element binding protein 5 and modulating hepatocytes phenotype

    PubMed Central

    Zhang, Xiaoyong; Liu, Hongyan; Xie, Zhanglian; Deng, Wangyu; Wu, Chunchen; Qin, Bo; Hou, Jinlin; Lu, Mengji

    2016-01-01

    Cellular microRNAs (miRNAs) are able to influence hepatitis B virus (HBV) replication directly by binding to HBV transcripts or indirectly by targeting cellular factors. Here, we investigate the effect of epigenetically regulated miR-449a on HBV replication and the underlying mechanisms. miR-449a expression was lower in human hepatocellular carcinoma (HCC) cells than in primary hepatocytes and could be induced by trichostatin A. Ectopic miR-449a expression in HCC cells strongly enhanced HBV replication, transcription, progeny virions secretion, and antigen expression in a dose-dependent manner. miR-449a directly targeted cAMP-responsive element binding protein 5 (CREB5), which in turn induced the expression of farnesoid X receptor α (FXRα), a transcription factor that facilitates HBV replication. CREB5 knockdown and overexpression demonstrated that it is a negative regulator of HBV replication. Additionally, miR-449a overexpression inhibited proliferation, caused cell cycle arrest, and promoted HCC cell differentiation. The results indicated that epigenetically regulated miR-449a targets CREB5 to increase FXRα expression, thereby promoting HBV replication and gene expression. Our findings provide a new understanding of the role of miRNAs in HBV replication. PMID:27138288

  20. IL-1 receptor antagonist (IL-1Ra) does not inhibit the production of C-reactive protein or serum amyloid A protein by human primary hepatocytes. Differential regulation in normal and tumour cells.

    PubMed Central

    Gabay, C; Genin, B; Mentha, G; Iynedjian, P B; Roux-Lombard, P; Guerne, P A

    1995-01-01

    The synthesis of some class 1 acute-phase proteins (APP), including C-reactive protein (CRP) and serum amyloid A (SAA) protein is completely blocked by the IL-1 receptor antagonist (IL-1Ra), whereas the production of fibrinogen, a class 2 APP, is increased by IL-1Ra in hepatoma cells, but this has never been tested in human hepatocytes in primary culture. Since previous studies on the contributions of cytokine inhibitors in connective tissues diseases suggested that IL-1 and tumour necrosis factor-alpha (TNF-alpha) might play an important role in the regulation of CRP, we decided to examine in more detail the respective roles of IL-1 beta, IL-6, and TNF-alpha and their inhibitors in the production of APP by human primary hepatocytes versus the hepatoma cell line PLC/PRF/5. In the hepatoma cell line, IL-1 beta and/or TNF-alpha had synergistic effects with IL-6 on the production of CRP and SAA. In contrast, these cytokines were devoid of effect in normal hepatocytes. The production of fibrinogen was increased by IL-6 and decreased by IL-1 (and TNF-alpha) in both cell types. The secretion of CRP and SAA by primary hepatocytes incubated with a cytokine-rich mononuclear cell-conditioned medium was totally unaffected by IL-1Ra or anti-TNF-alpha antibodies. In contrast, the addition of IL-1Ra increased the production of fibrinogen by both hepatoma cells and primary hepatocytes incubated with the mononuclear cell-conditioned medium. We therefore conclude that IL-1 beta and TNF-alpha do not exert any significant effect on the synthesis of CRP and SAA by human primary hepatocytes. Images Fig. 6 PMID:7743670

  1. Sho-saiko-to, a traditional herbal medicine, regulates gene expression and biological function by way of microRNAs in primary mouse hepatocytes

    PubMed Central

    2014-01-01

    Background Sho-saiko-to (SST) (also known as so-shi-ho-tang or xiao-chai-hu-tang) has been widely prescribed for chronic liver diseases in traditional Oriental medicine. Despite the substantial amount of clinical evidence for SST, its molecular mechanism has not been clearly identified at a genome-wide level. Methods By using a microarray, we analyzed the temporal changes of messenger RNA (mRNA) and microRNA expression in primary mouse hepatocytes after SST treatment. The pattern of genes regulated by SST was identified by using time-series microarray analysis. The biological function of genes was measured by pathway analysis. For the identification of the exact targets of the microRNAs, a permutation-based correlation method was implemented in which the temporal expression of mRNAs and microRNAs were integrated. The similarity of the promoter structure between temporally regulated genes was measured by analyzing the transcription factor binding sites in the promoter region. Results The SST-regulated gene expression had two major patterns: (1) a temporally up-regulated pattern (463 genes) and (2) a temporally down-regulated pattern (177 genes). The integration of the genes and microRNA demonstrated that 155 genes could be the targets of microRNAs from the temporally up-regulated pattern and 19 genes could be the targets of microRNAs from the temporally down-regulated pattern. The temporally up-regulated pattern by SST was associated with signaling pathways such as the cell cycle pathway, whereas the temporally down-regulated pattern included drug metabolism-related pathways and immune-related pathways. All these pathways could be possibly associated with liver regenerative activity of SST. Genes targeted by microRNA were moreover associated with different biological pathways from the genes not targeted by microRNA. An analysis of promoter similarity indicated that co-expressed genes after SST treatment were clustered into subgroups, depending on the temporal

  2. Novel Role for γ-Catenin in the Regulation of Cancer Cell Migration via the Induction of Hepatocyte Growth Factor Activator Inhibitor Type 1 (HAI-1)*

    PubMed Central

    Sechler, Marybeth; Borowicz, Stanley; Van Scoyk, Michelle; Avasarala, Sreedevi; Zerayesus, Sereke; Edwards, Michael G.; Kumar Karuppusamy Rathinam, Manoj; Zhao, Xiangmin; Wu, Pei-Ying; Tang, Ke; Bikkavilli, Rama Kamesh; Winn, Robert A.

    2015-01-01

    γ-catenin (Plakoglobin), a well-described structural protein functioning at the adherens junctions and desmosomes, was shown to be either lost or weakly expressed in non-small cell lung cancer (NSCLC) cells and tumor tissues. However, the tumor suppressive affects of γ-catenin were not fully understood. In this study, we have identified a novel role for the affects of γ-catenin on non-small cell lung cancer (NSCLC) cell migration. Expression of γ-catenin in NSCLC cells resulted in reduced cell migration as determined by both scratch assays and trans-well cell migration assays. Moreover, the affects of γ-catenin on cell migration were observed to be p53-dependent. Mechanistically, the anti-migratory effects seen via γ-catenin were driven by the expression of hepatocyte growth factor activator inhibitor Type I (HAI-1 or SPINT-1), an upstream inhibitor of the c-MET signaling pathway. Furthermore, the re-expression of γ-catenin sensitized NSCLC cells to c-MET inhibitor-mediated growth inhibition. Taken together, we identify γ-catenin as a novel regulator of HAI-1, which is a critical regulator of HGF/c-MET signaling. Therefore, targeting γ-catenin-mediated HAI-1 expression might be a useful strategy to sensitize NSCLC to c-MET inhibitors. PMID:25925948

  3. Insulin regulates the expression of several metabolism-related genes in the liver and primary hepatocytes of rainbow trout (Oncorhynchus mykiss).

    PubMed

    Plagnes-Juan, Elisabeth; Lansard, Marine; Seiliez, Iban; Médale, Françoise; Corraze, Geneviève; Kaushik, Sadasivam; Panserat, Stéphane; Skiba-Cassy, Sandrine

    2008-08-01

    Rainbow trout have a limited ability to use dietary carbohydrates efficiently and are considered to be glucose intolerant. Administration of carbohydrates results in persistent hyperglycemia and impairs post-prandial down regulation of gluconeogenesis despite normal insulin secretion. Since gluconeogenic genes are mainly under insulin control, we put forward the hypothesis that the transcriptional function of insulin as a whole may be impaired in the trout liver. In order to test this hypothesis, we performed intraperitoneal administration of bovine insulin to fasted rainbow trout and also subjected rainbow trout primary hepatocytes to insulin and/or glucose stimulation. We demonstrate that insulin was able to activate Akt, a key element in the insulin signaling pathway, and to regulate hepatic metabolism-related target genes both in vivo and in vitro. In the same way as in mammals, insulin decreased mRNA expression of gluconeogenic genes, including glucose 6-phosphatase (G6Pase), fructose 1,6-bisphosphatase (FBPase) and phosphoenolpyruvate carboxykinase (PEPCK). Insulin also limited the expression of carnitine palmitoyltransferase 1 (CPT1), a limiting enzyme of fatty acid beta-oxidation. In vitro studies revealed that, as in mammals, glucose is an important regulator of some insulin target genes such as the glycolytic enzyme pyruvate kinase (PK) and the lipogenic enzyme fatty acid synthase (FAS). Interestingly, glucose also stimulates expression of glucokinase (GK), which has no equivalent in mammals. This study demonstrates that insulin possesses the intrinsic ability to regulate hepatic gene expression in rainbow trout, suggesting that other hormonal or metabolic factors may counteract some of the post-prandial actions of insulin. PMID:18626086

  4. Distributed optical proximity sensor system - HexEYE

    NASA Technical Reports Server (NTRS)

    Lee, Sukhan

    1992-01-01

    A novel optical proximity sensor system, capable of measuring the distance to the orientation and the discontinuity at a local area of an object surface, was designed and evaluated for robotic applications. The sensor system gets its name, Hexagonal Eye, (HexEYE) from its shape where seven identical hexagonal sensing units were configured hexagonally into a compact geometric structure. The seven sensing units were grouped into multiple combinations of three sensing units to measure the surface orientation as well as surface discontinuity. The distinctive features of HexEYE are an order of magnitude increased in distance sensitivity by optimizing the curvature of a conical mirror, the enhanced measurement accuracy based on multiple levels of sensor fusion, and the compactness in size due to a sensing mechanism based on the Gaussian lens law. A prototype of single sensing unit has been built and was evaluated experimentally.

  5. SAMe Prevents the Up Regulation of Toll-Like Receptor Signaling in Mallory-Denk Body Forming Hepatocytes

    PubMed Central

    Bardag-Gorce, Fawzia; Oliva, Joan; Lin, Andrew; Li, Jun; French, Barbara A.; French, Samuel W.

    2010-01-01

    Mallory-Denk body (MDB) formation is a component of alcoholic and non alcoholic hepatitis. In the present study, the role of the toll-like receptor (TLR) signaling pathway was investigated in the mechanism of MDB formation in the DDC-fed mouse model. Microarray analysis data mining, performed on the livers of drug primed mice refed DDC, showed that TLR2/4 gene expression was significantly up regulated by DDC refeeding. SAMe supplementation prevented this up regulation and prevented the formation of MDBs. qRT-PCR analysis confirmed these results. TLR2/4 activates the adapter protein MyD88. The levels of MyD88 were increased by DDC refeeding. The increase of MyD88 was also prevented by SAMe supplementation. Results showed that MyD88-independent TLR3/4-TRIF-IRF3 pathway was not up regulated in the liver of DDC refed mice. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is the down stream protein recruited by the MyD88/IRAK protein complex, and is involved in the regulation of innate immune responses. Results showed a significant increase in the levels of TRAF-6. TRAF-6 activation leads to activation of NFkB and the mitogen-activated protein kinase (MAPK) cascade. The TRAF-6 increase was ameliorated by SAMe supplementation. These results suggest that DDC induces MDB formation through the TLR2/4 and MyD88-dependent signaling pathway. In conclusion, SAMe blocked the over-expression of TLR2/4, and their downstream signaling components MyD88 and TRAF-6. SAMe prevented the DDC-induced up regulation of the TLR signaling pathways, probably by preventing the up regulation of INF-γ receptors by DDC feeding. INFγ stimulates the up regulation of TLR2. The ability of SAMe feeding to prevent TLR signaling up regulation has not been previously described. PMID:20206621

  6. Downregulation of hepatocyte nuclear factor-4{alpha} and its role in regulation of gene expression by TGF-{beta} in mammary epithelial cells

    SciTech Connect

    Ishikawa, Fumihiro; Nose, Kiyoshi; Shibanuma, Motoko

    2008-06-10

    We found that a specific isoform of hepatocyte nuclear factor 4{alpha} (HNF-4{alpha}), HNF-4{alpha}8, was expressed in mouse mammary epithelial NMuMG cells, and that its expression was repressed by TGF-{beta}. The repression was interfered by dominant negative forms of activin receptor-like kinase 5 (ALK5) and Smad3, and sensitive to cycloheximide, suggesting the involvement of additional protein(s) as well as ALK5 and Smad3 in the repression. Further study showed that high mobility group A2 (HMGA2), which is reported to be directly upregulated by Smads, repressed HNF-4{alpha}8 expression. Therefore, it is likely that HMGA2 mediates the downregulation of HNF-4{alpha}8 downstream of ALK5 and Smads To determine the significance of the downregulation of HNF-4{alpha}8 in TGF-{beta} signaling, we performed DNA microarray analysis and extracted a subgroup of TGF-{beta}1-regulated genes, including tenascin C and tissue inhibitor of metalloproteinase 3 (TIMP-3), whose regulation by TGF-{beta}1 was attenuated by forced expression of HNF-4{alpha}8. HMGA2 has recently emerged as a transcriptional organizer of TGF-{beta} signaling, regulating several key factors involved in epithelial-mesenchymal transition (EMT). In this study, we identified an isoform of HNF-4{alpha} as a new target downstream of HMGA2 and assigned a new role to HNF-4{alpha} in the TGF-{beta} signaling/transcriptional cascade driven by ALK5/Smad/HMGA2 and associated with the malignant transformation of cells.

  7. Murine fumarylacetoacetate hydrolase (Fah) gene is disrupted by a neonatally lethal albino deletion that defines the hepatocyte-specific developmental regulation 1 (hsdr-1) locus

    SciTech Connect

    Klebig, M.L. Oak Ridge National Lab., TN ); Russell, L.B.; Rinchik, E.M. )

    1992-02-15

    Homozygous deletion of the hepatocyte-specific developmental regulation 1 (hsdr-1) locus in mouse chromosome 7 results in perinatal death and a pleiotropic syndrome characterized by ultrastructural abnormalities of the liver and kidney, failure of induction of a number of specific transcription units in the liver and kidney during late gestation, and marked overexpression of an enzyme that defends against oxidative stress. Previously, the breakpoints of two albino (c) deletions (c{sup 14CoS} and c{sup IFAFyh}) that genetically define hsdr-1 were localized, on a long-range map, in the vicinity of the distal breakpoint of a viable albino deletion (c{sup 24R75M}) that breaks proximally within the c locus. Here the authors report the use of a probe derived from a deletion breakpoint fusion fragment cloned from c{sup 24R75M}/c{sup 24R75M} DNA to clone a breakpoint fusion fragment caused by the c{sup 14CoS} deletion. The proximal breakpoint of the c{sup 14CoS} deletion was discovered to disrupt a gene (Fah) encoding fumarylacetoacetate hydrolase, the last enzyme in the tyrosine degradation pathway. These mouse mutants may also provide models for the human genetic disorder hereditary tyrosinemia, which is associated with fumarylacetoacetate hydrolase deficiency and liver and kidney dysfunction.

  8. Transcriptional regulation of the hepatocyte growth factor gene by the nuclear receptors chicken ovalbumin upstream promoter transcription factor and estrogen receptor.

    PubMed

    Jiang, J G; Bell, A; Liu, Y; Zarnegar, R

    1997-02-14

    Hepatocyte growth factor (HGF) is a multifunctional cytokine that controls the growth and differentiation of various tissues. Previously, we described the existence of a negative cis-acting regulatory element(s) within the -1- to -0.7-kilobase pair (kb) portion of the 5'-flanking region of the mouse HGF promoter. In the present study, we show that the repressor element is located at position -872 to -860 base pairs and comprises an imperfect estrogen-responsive element 5'-AGGTCAGAAAGACCA-3'. We demonstrate that chicken ovalbumin upstream promoter transcription factor (COUP-TF), a nuclear orphan receptor belonging to the steroid/thyroid hormone receptor superfamily, through binding to this site effectively silences the transcriptional activity of the HGF promoter. We show that estrogen receptor, on the other hand, relieves the repressive action of COUP-TF, resulting in the induction of the HGF promoter. Using mice transgenic for either 2.7 or 0.7 kb of the HGF promoter region linked to the chloramphenicol acetyltransferase reporter gene, we found that injection of estradiol stimulates HGF promoter activity in tissues such as the mammary gland and ovary of mice harboring 2.7 but not 0.7 kb of the mouse HGF promoter region. Potential involvement of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors in the regulation of HGF gene expression is also discussed. PMID:9020096

  9. Transcriptional Regulation of X-Box-binding Protein One (XBP1) by Hepatocyte Nuclear Factor 4α (HNF4Α) Is Vital to Beta-cell Function.

    PubMed

    Moore, Benjamin D; Jin, Ramon U; Lo, Heiyong; Jung, Min; Wang, Haiyan; Battle, Michele A; Wollheim, Claes B; Urano, Fumihiko; Mills, Jason C

    2016-03-18

    The transcription factor, X-box-binding protein-1 (XBP1), controls the development and maintenance of the endoplasmic reticulum (ER) in multiple secretory cell lineages. We show here that Hepatocyte Nuclear Factor 4α (HNF4α) directly induces XBP1 expression. Mutations in HNF4α cause Mature-Onset Diabetes of the Young I (MODYI), a subset of diabetes characterized by diminished GSIS. In mouse models, cell lines, and ex vivo islets, using dominant negative and human- disease-allele point mutants or knock-out and knockdown models, we show that disruption of HNF4α caused decreased expression of XBP1 and reduced cellular ER networks. GSIS depends on ER Ca(2+) signaling; we show that diminished XBP1 and/or HNF4α in β-cells led to impaired ER Ca(2+) homeostasis. Restoring XBP1 expression was sufficient to completely rescue GSIS in HNF4α-deficient β-cells. Our findings uncover a transcriptional relationship between HNF4α and Xbp1 with potentially broader implications about MODYI and the importance of transcription factor signaling in the regulation of secretion. PMID:26792861

  10. Hepatocyte growth factor inhibits apoptosis by the profibrotic factor angiotensin II via extracellular signal-regulated kinase 1/2 in endothelial cells and tissue explants.

    PubMed

    Lee, Young H; Marquez, Ana P; Mungunsukh, Ognoon; Day, Regina M

    2010-12-01

    Hepatocyte growth factor (HGF), an endogenous tissue repair factor, attenuates apoptosis in many primary cell types, but the mechanism is not completely understood. Our laboratory demonstrated that angiotensin (Ang) II activates the intrinsic apoptotic pathway in primary endothelial cells (ECs) via reduction of the antiapoptotic protein Bcl-x(L). Ang II decreased Bcl-x(L) mRNA half-life by reducing its binding to nucleolin, a protein that normally binds a 3' AU-rich region and stabilizes Bcl-x(L) mRNA. We hypothesized HGF may block apoptosis induced by Ang II. We used primary EC and ex vivo cultures of rat lung tissue to investigate HGF inhibition of Ang II-induced apoptosis. Our data indicated HGF abrogated Ang II-induced apoptosis by inhibiting cytochrome c release, caspase-3 activation, and DNA fragmentation. RNA-immunoprecipitation experiments demonstrated that HGF stabilized Bcl-x(L) mRNA by increasing nucleolin binding to the 3'-untranslated region that was associated with cytoplasmic localization of nucleolin. Cytoplasmic localization of nucleolin and Bcl-x(L) mRNA stabilization required HGF activation of extracellular signal-regulated kinase (ERK)1/2, but not phosphatidylinositol 3-kinase. HGF also blocked Ang II-induced caspase-3 activation and lactate dehydrogenase release in tissue explants in an ERK-dependent manner. PMID:20926686

  11. Hepatocyte nuclear factor 1 regulates the expression of the organic cation transporter 1 via binding to an evolutionary conserved region in intron 1 of the OCT1 gene.

    PubMed

    O'Brien, Valerie P; Bokelmann, Kristin; Ramírez, Jacqueline; Jobst, Karoline; Ratain, Mark J; Brockmöller, Jürgen; Tzvetkov, Mladen V

    2013-10-01

    The organic cation transporter 1 (OCT1), also known as solute carrier family 22 member 1, is strongly and specifically expressed in the human liver. Here we show that the hepatocyte nuclear factor 1 (HNF1) regulates OCT1 transcription and contributes to the strong, liver-specific expression of OCT1. Bioinformatic analyses revealed strong conservation of HNF1 binding motifs in an evolutionary conserved region (ECR) in intron 1 of the OCT1 gene. Electrophoretic mobility shift and chromatin immunoprecipitation assays confirmed the specific binding of HNF1 to the intron 1 ECR. In reporter gene assays performed in HepG2 cells, the intron 1 ECR increased SV40 promoter activity by 22-fold and OCT1 promoter activity by 13-fold. The increase was reversed when the HNF1 binding sites in the intron 1 ECR were mutated or the endogenous HNF1α expression was downregulated with small interfering RNA. Following HNF1α overexpression in Huh7 cells, the intron 1 ECR increased SV40 promoter activity by 11-fold and OCT1 promoter activity by 6-fold. Without HNF1α overexpression, the increases were only 3- and 2-fold, respectively. Finally, in human liver samples, high HNF1 expression was significantly correlated with high OCT1 expression (r = 0.48, P = 0.002, n = 40). In conclusion, HNF1 is a strong regulator of OCT1 expression. It remains to be determined whether genetic variants, disease conditions, or drugs that affect HNF1 activity may affect the pharmacokinetics and efficacy of OCT1-transported drugs such as morphine, tropisetron, ondansetron, tramadol, and metformin. Beyond OCT1, this study demonstrates the validity and usefulness of interspecies comparisons in the discovery of functionally relevant genomic sequences. PMID:23922447

  12. Hepatocyte Nuclear Factor 1 Regulates the Expression of the Organic Cation Transporter 1 via Binding to an Evolutionary Conserved Region in Intron 1 of the OCT1 Gene

    PubMed Central

    O’Brien, Valerie P.; Bokelmann, Kristin; Ramírez, Jacqueline; Jobst, Karoline; Ratain, Mark J.; Brockmöller, Jürgen

    2013-01-01

    The organic cation transporter 1 (OCT1), also known as solute carrier family 22 member 1, is strongly and specifically expressed in the human liver. Here we show that the hepatocyte nuclear factor 1 (HNF1) regulates OCT1 transcription and contributes to the strong, liver-specific expression of OCT1. Bioinformatic analyses revealed strong conservation of HNF1 binding motifs in an evolutionary conserved region (ECR) in intron 1 of the OCT1 gene. Electrophoretic mobility shift and chromatin immunoprecipitation assays confirmed the specific binding of HNF1 to the intron 1 ECR. In reporter gene assays performed in HepG2 cells, the intron 1 ECR increased SV40 promoter activity by 22-fold and OCT1 promoter activity by 13-fold. The increase was reversed when the HNF1 binding sites in the intron 1 ECR were mutated or the endogenous HNF1α expression was downregulated with small interfering RNA. Following HNF1α overexpression in Huh7 cells, the intron 1 ECR increased SV40 promoter activity by 11-fold and OCT1 promoter activity by 6-fold. Without HNF1α overexpression, the increases were only 3- and 2-fold, respectively. Finally, in human liver samples, high HNF1 expression was significantly correlated with high OCT1 expression (r = 0.48, P = 0.002, n = 40). In conclusion, HNF1 is a strong regulator of OCT1 expression. It remains to be determined whether genetic variants, disease conditions, or drugs that affect HNF1 activity may affect the pharmacokinetics and efficacy of OCT1-transported drugs such as morphine, tropisetron, ondansetron, tramadol, and metformin. Beyond OCT1, this study demonstrates the validity and usefulness of interspecies comparisons in the discovery of functionally relevant genomic sequences. PMID:23922447

  13. Physiological Expression and Accumulation of the Products of Two Upstream Open Reading Frames mrtl and MycHex1 Along With p64 and p67 Myc From the Human c-myc Locus.

    PubMed

    Ji, Mi Hong; Kim, Seung-Ki; Kim, Chae-Yong; Phi, Ji Hoon; Jun, Hyun Jin; Blume, Scott W; Choi, Hyoung Soo

    2016-06-01

    In addition to the canonical c-Myc p64 and p67 proteins, the human c-myc locus encodes two distinct proteins, mrtl (myc-related translation/localization regulatory factor) and MycHex1 (Myc Human Exon 1), from the upstream open reading frames within the 5'-untranslated region of the c-myc P0 mRNA. The aim of this study is to examine simultaneously, for the first time, mrtl, MycHex1, c-Myc p64, and p67 in human tumor cell lines and pediatric brain tumor tissues. Western blot analysis demonstrated endogenous mrtl, MycHex1, c-Myc p64, and p67 simultaneously. The relative abundance of mrtl and MycHex1 were consistent among a variety of human tumor cell lines, and the relative intensities of mrtl and MycHex1 correlated positively. Confocal imaging revealed mrtl predominantly localized to the nuclear envelope, along with prominent reticular pattern in the cytoplasm. MycHex1 was observed as a series of bright foci located within the nucleus, a subset of which colocalized with fibrillarin. mrtl and MycHex1 co-immunoprecipitated with RACK1, c-Myc, fibrillarin, coilin, and with each other. These findings suggest that mrtl and MycHex1 have multiple interaction partners in both the nucleus and cytoplasm. Sequence analyses confirmed a known polymorphism of mrtl at base 1965 (G>T) and new mutations at bases 1900 (C>G) and 1798 (C>G). Evidence is presented for expression and stable accumulation of all four proteins encoded by three distinct non-overlapping open reading frames within the human c-myc locus. Additional work is warranted to further elucidate the functional or regulatory roles of these molecules in regulation of c-Myc and in oncogenesis. J. Cell. Biochem. 117: 1407-1418, 2016. © 2015 Wiley Periodicals, Inc. PMID:26552949

  14. Heparin-binding growth factor type 1 (acidic fibroblast growth factor): a potential biphasic autocrine and paracrine regulator of hepatocyte regeneration.

    PubMed Central

    Kan, M; Huang, J S; Mansson, P E; Yasumitsu, H; Carr, B; McKeehan, W L

    1989-01-01

    Heparin-binding growth factor type 1 (HBGF-1; sometimes termed acidic fibroblast growth factor) is potentially an important factor in liver regeneration. HBGF-1 alone (half-maximal effect at 60 pM) stimulated hepatocyte DNA synthesis and bound to a high-affinity receptor (Kd = 62 pM; 5000 per cell). Epidermal growth factor (EGF) neutralized or masked the mitogenic effect of HBGF-1 concurrent with appearance of low-affinity HBGF-1 binding sites. HBGF-1 reduced the inhibitory effect of transforming growth factor type beta (TGF-beta) on the EGF stimulus. Nanomolar levels of HBGF-1 decreased the EGF stimulus. An increase in hepatic HBGF-1 gene expression after partial hepatectomy precedes increases in expression of the EGF homolog, TGF-alpha, and nonparenchymal-cell-derived TGF-beta in the regenerating liver. Expression of HBGF-1 mRNA occurs in both hepatocytes and nonparenchymal cells and persists for 7 days in liver tissue after partial hepatectomy. HBGF-1 acting through a high-affinity receptor is a candidate for the early autocrine stimulus that drives hepatocyte DNA synthesis prior to or concurrent with the EGF/TGF-alpha stimulus. It may allow hepatocyte proliferation to proceed in the presence of low levels of TGF-beta. An EGF/TGF-alpha-dependent change in HBGF-1 receptor phenotype and increasing levels of nonparenchymal-cell-derived HBGF-1 and TGF-beta may serve to limit hepatocyte proliferation. Images PMID:2477840

  15. Ascorbic acid partly antagonizes resveratrol mediated heme oxygenase-1 but not paraoxonase-1 induction in cultured hepatocytes - role of the redox-regulated transcription factor Nrf2

    PubMed Central

    2011-01-01

    Background Both resveratrol and vitamin C (ascorbic acid) are frequently used in complementary and alternative medicine. However, little is known about the underlying mechanisms for potential health benefits of resveratrol and its interactions with ascorbic acid. Methods The antioxidant enzymes heme oxygenase-1 and paraoxonase-1 were analysed for their mRNA and protein levels in HUH7 liver cells treated with 10 and 25 μmol/l resveratrol in the absence and presence of 100 and 1000 μmol/l ascorbic acid. Additionally the transactivation of the transcription factor Nrf2 and paraoxonase-1 were determined by reporter gene assays. Results Here, we demonstrate that resveratrol induces the antioxidant enzymes heme oxygenase-1 and paraoxonase-1 in cultured hepatocytes. Heme oxygenase-1 induction by resveratrol was accompanied by an increase in Nrf2 transactivation. Resveratrol mediated Nrf2 transactivation as well as heme oxygenase-1 induction were partly antagonized by 1000 μmol/l ascorbic acid. Conclusions Unlike heme oxygenase-1 (which is highly regulated by Nrf2) paraoxonase-1 (which exhibits fewer ARE/Nrf2 binding sites in its promoter) induction by resveratrol was not counteracted by ascorbic acid. Addition of resveratrol to the cell culture medium produced relatively low levels of hydrogen peroxide which may be a positive hormetic redox-signal for Nrf2 dependent gene expression thereby driving heme oxygenase-1 induction. However, high concentrations of ascorbic acid manifold increased hydrogen peroxide production in the cell culture medium which may be a stress signal thereby disrupting the Nrf2 signalling pathway. PMID:21199573

  16. Hepatocyte Growth Factor Activator Inhibitor-1 Is Induced by Bone Morphogenetic Proteins and Regulates Proliferation and Cell Fate of Neural Progenitor Cells

    PubMed Central

    Koivuniemi, Raili; Mäkelä, Johanna; Hokkanen, Marie-Estelle; Bruelle, Céline; Ho, Tho Huu; Ola, Roxana; Korhonen, Laura; Schröder, Jim; Kataoka, Hiroaki; Lindholm, Dan

    2013-01-01

    Background Neural progenitor cells (NPCs) in the developing neuroepithelium are regulated by intrinsic and extrinsic factors. There is evidence that NPCs form a self-supporting niche for cell maintenance and proliferation. However, molecular interactions and cell-cell contacts and the microenvironment within the neuroepithelium are largely unknown. We hypothesized that cellular proteases especially those associated with the cell surface of NPCs play a role in regulation of progenitor cells in the brain. Methodology/Principal Findings In this work, we show that NPCs, isolated from striatal anlage of developing rat brain, express hepatocyte growth factor activator inhibitor-1 and -2 (HAI-1 and HAI-2) that are cell surface-linked serine protease inhibitors. In addition, radial glia cells derived from mouse embryonic stem cells also express HAI-1 and HAI-2. To study the functional significance of HAI-1 and HAI-2 in progenitor cells, we modulated their levels using expression plasmids or silencing RNA (siRNA) transfected into the NPCs. Data showed that overexpression of HAI-1 or HAI-2 decreased cell proliferation of cultured NPCs, whilst their siRNAs had opposite effects. HAI-1 also influenced NPC differentiation by increasing the number of glial fibrillary acidic protein (GFAP) expressing cells in the culture. Expression of HAI-1 in vivo decreased cell proliferation in developing neuroepithelium in E15 old animals and promoted astrocyte cell differentiation in neonatal animals. Studying the regulation of HAI-1, we observed that Bone morphogenetic protein-2 (BMP-2) and BMP-4 increased HAI-1 levels in the NPCs. Experiments using HAI-1-siRNA showed that these BMPs act on the NPCs partly in a HAI-1-dependent manner. Conclusions This study shows that the cell-surface serine protease inhibitors, HAI-1 and HAI-2 influence proliferation and cell fate of NPCs and their expression levels are linked to BMP signaling. Modulation of the levels and actions of HAI-1 in NPCs may be of

  17. Hex-dominant mesh generation using 3D constrained triangulation

    SciTech Connect

    OWEN,STEVEN J.

    2000-05-30

    A method for decomposing a volume with a prescribed quadrilateral surface mesh, into a hexahedral-dominated mesh is proposed. With this method, known as Hex-Morphing (H-Morph), an initial tetrahedral mesh is provided. Tetrahedral are transformed and combined starting from the boundary and working towards the interior of the volume. The quadrilateral faces of the hexahedra are treated as internal surfaces, which can be recovered using constrained triangulation techniques. Implementation details of the edge and face recovery process are included. Examples and performance of the H-Morph algorithm are also presented.

  18. Hex-Sided Rounded Dipole Antenna (HSRDA) For UWB Applications

    NASA Astrophysics Data System (ADS)

    Singhal, Sarthak; Verma, Nand Kishor; Singh, Amit Kumar

    2016-03-01

    A hex-sided rounded dipole antenna (HSRDA) for UWB applications is presented. It is designed by the addition of semi-elliptical patch sections at the edges of a square bow-tie antenna. The antenna structure is fed by a modified microstrip feedline for better impedance matching. An impedance bandwidth of 2.9-11.4 GHz is achieved. The antenna structure has quasi omnidirectional radiation patterns and reasonable gain over the same frequency range. A good agreement between the experimental and simulation results is observed. The proposed antenna structure has miniaturized size for the same bandwidth as compared to already reported antenna structures.

  19. Exploring the cell signalling in hepatocyte differentiation.

    PubMed

    Vasconcellos, Rebecca; Alvarenga, Érika C; Parreira, Ricardo C; Lima, Swiany S; Resende, Rodrigo R

    2016-11-01

    The liver is the second largest organ in the human body and is responsible for several functions that directly contribute to homeostasis. Hepatocytes are the main parenchymal liver cells that regulate multiple biochemical and metabolic functions and the synthesis of substances important to the body. Mesenchymal stem cells (MSCs) are a group of stem cells derived from the mesoderm, which can be obtained from various tissues. Under certain conditions, MSCs can differentiate into several cell types, including hepatocytes. Post-transcriptional regulations of liver development signalling and hepatocyte differentiation have been demonstrated. At the post-transcriptional level, microRNAs have emerged as precursors for determining cell fate during differentiation. MicroRNAs (miRNAs) are small non-coding RNAs involved in the post-transcriptional regulation of gene expression. They can determine the stem cell fate by repressing the translation of target mRNAs. In this review, we outline signalling pathways involved in stem cell differentiation to hepatocytes and its interplay with liver development. Hepatic differentiation models in two-dimensional and three-dimensional cultures used to analyse signalling mechanisms will be described. We also highlight the possible miRNAs involved in this process and the transdifferentiation signalling mechanisms present in hepatocytes. PMID:27555287

  20. HexSim - A general purpose framework for spatially-explicit, individual-based modeling

    EPA Science Inventory

    HexSim is a framework for constructing spatially-explicit, individual-based computer models designed for simulating terrestrial wildlife population dynamics and interactions. HexSim is useful for a broad set of modeling applications. This talk will focus on a subset of those ap...

  1. In vivo role of Candida albicans β-hexosaminidase (HEX1) in carbon scavenging

    PubMed Central

    Ruhela, Deepa; Kamthan, Mohan; Saha, Paramita; Majumdar, Subeer S; Datta, Kasturi; Abdin, Malik Zainul; Datta, Asis

    2015-01-01

    The capability to utilize of N-acetylglucosamine (GlcNAc) as a carbon source is an important virulence attribute of Candida albicans. But there is a lack of information about the in vivo source of GlcNAc for the pathogen within the host environment. Here, we have characterized the GlcNAc-inducible β-hexosaminidase gene (HEX1) of C. albicans showing a role in carbon scavenging. In contrast to earlier studies, we have reported HEX1 to be a nonessential gene as shown by homozygous trisomy test. Virulence study in the systemic mouse murine model showed that Δhex1 strain is significantly less virulent in comparison to the wild-type strain. Moreover, Δhex1 strain also showed a higher susceptibility to peritoneal macrophages. In an attempt to determine possible substrates of Hex1, hyaluronic acid (HA) was treated with purified Hex1 enzyme. A significant release of GlcNAc was observed by gas chromatography-mass spectrometry analysis analysis suggesting HA degradation. Interestingly, immunohistochemistry analysis showed significant accumulation of HA in the mice kidney infected with the wild-type strain of C. albicans. Northern blot analysis showed that C. albicans HEX1 is expressed during mice renal colonization. Thus, C. albicans can obtain GlcNAc during organ colonization by secreting Hex1 via degradation of host HA. PMID:26177944

  2. Hepatocytes as Immunological Agents.

    PubMed

    Crispe, Ian N

    2016-01-01

    Hepatocytes are targeted for infection by a number of major human pathogens, including hepatitis B virus, hepatitis C virus, and malaria. However, hepatocytes are also immunological agents in their own right. In systemic immunity, they are central in the acute-phase response, which floods the circulation with defensive proteins during diverse stresses, including ischemia, physical trauma, and sepsis. Hepatocytes express a variety of innate immune receptors and, when challenged with pathogen- or damage-associated molecular patterns, can deliver cell-autonomous innate immune responses that may result in host defense or in immunopathology. Important human pathogens have evolved mechanisms to subvert these responses. Finally, hepatocytes talk directly to T cells, resulting in a bias toward immune tolerance. PMID:26685314

  3. The Salivary β-HEX A% Index as an Excellent Marker of Periodontitis in Smoking Alcohol-Dependent Persons

    PubMed Central

    Waszkiewicz, Napoleon; Zalewska-Szajda, Beata; Chojnowska, Sylwia; Szajda, Sławomir Dariusz; Zalewska, Anna; Konarzewska, Beata; Szulc, Agata; Wojtulewska-Supron, Aleksandra; Kępka, Alina; Knaś, Małgorzata; Ładny, Jerzy Robert; Milewski, Robert; Zwierz, Krzysztof

    2013-01-01

    Background. Severe periodontitis leading to tooth loss is found in 5–15% of most populations worldwide. Aim. The applicability of salivary β-hexosaminidase (β-HEX A%, percentage of β-HEX A isoenzyme to total β-HEX) and β-HEX B% (β-HEX B/β-HEX) indexes was investigated as a possible marker of periodontitis. Methods. Thirty three alcohol-dependent smokers (AS) and 32 healthy controls (C) were enrolled in the study. The activity of β-HEX was measured spectrophotometrically. Results. β-HEX A% was significantly higher and β-HEX B% was lower in AS than in C group. We found a significant correlation between β-HEX A% and gingival index (GI) and an inverse correlation between β-HEX A% and salivary flow (SF), in all groups. Salivary β-HEX A% index in smoking alcoholics at 0.23 had excellent sensitivity (96%) and specificity (91%); the AUC for β-HEX A% was high (0.937). There were no correlations between amount/duration-time of alcohol drinking/smoking and β-HEX A% or β-HEX B%. We found significant correlations between the time period of denture wearing and GI, papilla bleeding index (PBI), and decayed missing filled teeth index (DMFT) and between GI and the amount of smoked cigarettes per day. Conclusion. Bad periodontal state was most likely due to the nicotine dependence. Salivary β-HEX A% is a promising excellent marker for the diagnosis of periodontitis. PMID:24288426

  4. In Vitro Culture of Functionally Active Buffalo Hepatocytes Isolated by Using a Simplified Manual Perfusion Method

    PubMed Central

    Panda, Santanu; Bisht, Sonu; Malakar, Dhruba; Mohanty, Ashok K.; Kaushik, Jai K.

    2015-01-01

    Background In farm animals, there is no suitable cell line available to understand liver-specific functions. This has limited our understanding of liver function and metabolism in farm animals. Culturing and maintenance of functionally active hepatocytes is difficult, since they survive no more than few days. Establishing primary culture of hepatocytes can help in studying cellular metabolism, drug toxicity, hepatocyte specific gene function and regulation. Here we provide a simple in vitro method for isolation and short-term culture of functionally active buffalo hepatocytes. Results Buffalo hepatocytes were isolated from caudate lobes by using manual enzymatic perfusion and mechanical disruption of liver tissue. Hepatocyte yield was (5.3±0.66)×107 cells per gram of liver tissue with a viability of 82.3±3.5%. Freshly isolated hepatocytes were spherical with well contrasted border. After 24 hours of seeding onto fibroblast feeder layer and different extracellular matrices like dry collagen, matrigel and sandwich collagen coated plates, hepatocytes formed confluent monolayer with frequent clusters. Cultured hepatocytes exhibited typical cuboidal and polygonal shape with restored cellular polarity. Cells expressed hepatocyte-specific marker genes or proteins like albumin, hepatocyte nuclear factor 4α, glucose-6-phosphatase, tyrosine aminotransferase, cytochromes, cytokeratin and α1-antitrypsin. Hepatocytes could be immunostained with anti-cytokeratins, anti-albumin and anti α1-antitrypsin antibodies. Abundant lipid droplets were detected in the cytosol of hepatocytes using oil red stain. In vitro cultured hepatocytes could be grown for five days and maintained for up to nine days on buffalo skin fibroblast feeder layer. Cultured hepatocytes were viable for functional studies. Conclusion We developed a convenient and cost effective technique for hepatocytes isolation for short-term culture that exhibited morphological and functional characteristics of active

  5. NKP30-B7-H6 Interaction Aggravates Hepatocyte Damage through Up-Regulation of Interleukin-32 Expression in Hepatitis B Virus-Related Acute-On-Chronic Liver Failure

    PubMed Central

    Pan, Xingfei; Lu, Ying; Liao, Sihong; Wang, Xicheng; Wang, Guoying; Lin, Dongjun

    2015-01-01

    Background and Aims Previous work conducted by our group has shown that the accumulation of hepatic natural killer (NK) cells and the up-regulation of natural cytotoxicity receptors (NKP30 and NKP46) on NK cells from patients with hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) were correlated with disease progression in HBV-ACLF. The natural cytotoxicity receptors expressed on NK cells are believed to be probable candidates involved in the NK cell-mediated hepatocyte damage in HBV-ACLF. However, the underlying mechanisms remain to be elucidated. In the present study, we aimed to discover the role of NKP30-B7-H6 interaction in NK cells-mediated hepatocyte damage in HBV-ACLF. Methods Hepatic expressions of B7-H6 and interleukin-32 (IL-32) were examined by immunochemistry staining in samples from patients with HBV-ACLF or mild chronic hepatitis B (CHB). The cytotoxicity of NK-92 cell against target cells (Huh-7 and LO2) was evaluated by CCK8 assay. Expression of IL-32 in liver NK cell, T cells and NK-92 cell line was detected by the flow cytometric analysis. The effect of IL-32 on the apoptosis of Huh7 cells was evaluated using Annexin V/PI staining analysis. Results An enhancement of hepatic B7-H6 and IL-32 expression was associated with the severity of liver injury in HBV-ACLF. And there was a positive association between hepatic B7-H6 and IL-32 expression. Expressions of IL-32 in liver NK cells and T cells were increased in HBV-ACLF patients. In vitro NK-92 cells are highly capable of killing the high B7-H6 expressing Huh7 cells and B7-H6-tansfected hepatocyte line LO2 cells dependent on NKP30 and B7-H6 interaction. Furthermore, NK-92 cells exhibited elevated IL-32 expression when stimulated with anti-NKP30 antibodies or when co-cultured with Huh7 cells. IL-32 can induce the apoptosis of Huh7 cells in a dose-dependent manner. Conclusion Our results suggest that NKP30-B7-H6 interaction can aggravate hepatocyte damage, probably through up-regulation

  6. Suppression of Hepatocyte Proliferation by Hepatocyte Nuclear Factor 4α in Adult Mice*

    PubMed Central

    Bonzo, Jessica A.; Ferry, Christina H.; Matsubara, Tsutomu; Kim, Jung-Hwan; Gonzalez, Frank J.

    2012-01-01

    Hepatocyte nuclear factor 4α (HNF4α) regulates genes involved in lipid and bile acid synthesis, gluconeogenesis, amino acid metabolism, and blood coagulation. In addition to its metabolic role, HNF4α is critical for hepatocyte differentiation, and loss of HNF4α is associated with hepatocellular carcinoma. The hepatocyte-specific Hnf4a knock-out mouse develops severe hepatomegaly and steatosis resulting in premature death, thereby limiting studies of the role of this transcription factor in the adult animal. In addition, gene compensation may complicate analysis of the phenotype of these mice. To overcome these issues, an acute Hnf4a knock-out mouse model was generated through use of the tamoxifen-inducible ErT2cre coupled to the serum albumin gene promoter. Microarray expression analysis revealed up-regulation of genes associated with proliferation and cell cycle control only in the acute liver-specific Hnf4α-null mouse. BrdU and ki67 staining confirmed extensive hepatocyte proliferation in this model. Proliferation was associated with induction of the hepatomitogen Bmp7 as well as reduced basal apoptotic activity. The p53/p63 apoptosis effector gene Perp was further identified as a direct HNF4α target gene. These data suggest that HNF4α maintains hepatocyte differentiation in the adult healthy liver, and its loss may directly contribute to hepatocellular carcinoma development, thus indicating this factor as a possible liver tumor suppressor gene. PMID:22241473

  7. S phase-specific DNA-binding proteins interacting with the Hex and Oct motifs in type I element of the wheat histone H3 promoter.

    PubMed

    Minami, M; Meshi, T; Iwabuchi, M

    2000-01-11

    The type I element (CCACGTCANCGATCCGCG), consisting of the Hex motif (CCACGTCA) and the reverse-oriented Oct motif (GATCCGCG), is necessary and sufficient to confer the S phase-specific transcription of the wheat histone H3 (TH012) gene. The transcriptional regulation via the type I element is thought to occur through interactions between transcription factors which bind specifically to the Hex and Oct motifs. Here we report S phase-specific DNA-binding proteins interacting with the type I element in partially synchronized wheat cultured cells. Hex motif-binding proteins found here resembled HBP-1a, as reported previously in terms of DNA-binding specificity. DNA-binding activities of the HBP-1a-like proteins were modulated by phosphorylation/dephosphorylation. In the electrophoretic mobility shift assay of the wheat nuclear extract, we also found three Oct motif-specific binding proteins, named OBRF (octamer-binding regulatory factor)-1, -2 and -3. One of the HBP-1a-like proteins and OBRF-1 appeared predominantly at the S phase. Thus, it was supposed that these two factors play a crucial role in the S phase-specific regulation of wheat histone gene expression. PMID:10675046

  8. Bacterial contamination along implant-abutment interface in external and internal-hex dental implants

    PubMed Central

    de Oliveira, Greison Rabelo; Olate, Sergio; Pozzer, Leandro; Cavalieri-Pereira, Lucas; Rodrigues-Chessa, Jaime G; Albergaría-Barbosa, José Ricardo

    2014-01-01

    The aim of this research was to evaluate bacterial contamination along the implant-abutment interface in relation to the size of the interface. 80 brand name implants were used, 40 internal-hex and 40 external-hex. The implants were handled in a sterile atmosphere inside a box, where they were inoculated with 0.3 μl of the Streptococcus sanguis ATCC10556 bacterium in the interior and the abutment was immediately installed with a torque of 30 Ncm for the external-hex and 20 Ncm for the internal-hex; the system was included in an Eppendorf control for 30 seconds and then placed in an Eppendorf control for 30 days. The implants were removed and assessed under a scanning electron microscope while the Eppendorf controls were bred in blood agar to analyze the colonies formed. The data were analyzed using the Chi-squared, Kruskal-Wallis and Mann-Whitney tests, considering a value of p<0.05 to obtain statistical significance. Five implants were excluded due to probable external contamination. Microspaces of up to 86.8 μm were observed in the external-hex implants and up to 53.9 μm in the internal-hex implants with no significant differences between the different systems being observed (p>0.05). The contamination observed was produced mainly in the external-hex implants and statistically significant differences were observed between the different hex systems from the same company. No significant differences were observed between interface size and bacterial contamination. Within our limitations, there was no relation between the size of the implant-abutment interface and bacterial contamination with Streptococcus sanguis ATCC10556. PMID:24753751

  9. The impact of duct-to-duct interaction on the hex duct dilation

    SciTech Connect

    Lee, M.J.; Chang, L.K.; Lahm, C.E.; Porter, D.L.

    1992-07-01

    Dilation of the hex duct is an important factor in the operational lifetime of fuel subassemblies in liquid metal fast reactors. It is caused primarily by the irradiation-enhanced creep and void swelling of the hex duct material. Excessive dilation may jeopardize subassembly removal from the core or cause a subassembly storage problem where the grid size of the storage basket is limited. Dilation of the hex duct in Experimental Breeder Reactor II (EBR-II) limits useful lifetime because of these storage basket limitations. It is, therefore, important to understand the hex duct dilation behavior to guide the design and in-core management of fuel subassemblies in a way that excessive duct deformation can be avoided. To investigate the dilation phenomena, finite-element models of the hex duct have been developed. The inelastic analyses were performed using the structural analysis code, ANSYS. Both Type 316 and D9 austenitic stainless steel ducts are considered. The calculated dilations are in good agreement with profilometry measurements made after irradiation. The analysis indicates that subassembly interaction is an important parameter in addition to neutron fluence and temperature in determining hex duct dilation. 5 refs.

  10. Regulation of cytochrome P450 mRNA expression in primary porcine hepatocytes by selected secondary plant metabolites from chicory (Cichorium intybus L.).

    PubMed

    Rasmussen, Martin Krøyer; Klausen, Christina Lindgaard; Ekstrand, Bo

    2014-03-01

    Chicory (Cichorium intybus) has been shown to induce enzymes of pharmacokinetic relevance (cytochrome P450; CYP). The aim of this study was to investigate the effects of selected secondary plant metabolites with a global extract of chicory root, on the expression of hepatic CYP mRNA (1A2, 2A19, 2C33, 2D25, 2E1 and 3A29), using primary porcine hepatocytes. Of the tested secondary plant metabolites, artemisinin, scoparone, lactucin and esculetin all induced increased expression of specific CYPs, while esculin showed no effect. In contrast, a global extract of chicory root decreased the expression of CYP1A2, 2C33, 2D25 and 3A29 at high concentrations. The results suggest that purified secondary metabolites from chicory affect CYP expression and thereby might affect detoxification in general, and that global extracts of plants can have effects different from individual components. PMID:24176340

  11. Phytol directly activates peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) and regulates gene expression involved in lipid metabolism in PPAR{alpha}-expressing HepG2 hepatocytes

    SciTech Connect

    Goto, Tsuyoshi; Takahashi, Nobuyuki; Kato, Sota; Egawa, Kahori; Ebisu, Shogo; Moriyama, Tatsuya; Fushiki, Tohru; Kawada, Teruo . E-mail: fat@kais.kyoto-u.ac.jp

    2005-11-18

    The peroxisome proliferator-activated receptor (PPAR) is one of the indispensable transcription factors for regulating lipid metabolism in various tissues. In our screening for natural compounds that activate PPAR using luciferase assays, a branched-carbon-chain alcohol (a component of chlorophylls), phytol, has been identified as a PPAR{alpha}-specific activator. Phytol induced the increase in PPAR{alpha}-dependent luciferase activity and the degree of in vitro binding of a coactivator, SRC-1, to GST-PPAR{alpha}. Moreover, the addition of phytol upregulated the expression of PPAR{alpha}-target genes at both mRNA and protein levels in PPAR{alpha}-expressing HepG2 hepatocytes. These findings indicate that phytol is functional as a PPAR{alpha} ligand and that it stimulates the expression of PPAR{alpha}-target genes in intact cells. Because PPAR{alpha} activation enhances circulating lipid clearance, phytol may be important in managing abnormalities in lipid metabolism.

  12. Phase 2: HGM air flow tests in support of HEX vane investigation

    NASA Astrophysics Data System (ADS)

    Cox, G. B., Jr.; Steele, L. L.; Eisenhart, D. W.

    1993-07-01

    Following the start of SSME certification testing for the Pratt and Whitney Alternate Turbopump Development (ATD) High Pressure Oxidizer Turbopump (HPOTP), cracking of the leading edge of the inner HEX vane was experienced. The HEX vane, at the inlet of the oxidizer bowl in the Hot Gas Manifold (HGM), accepts the HPOTP turbine discharge flow and turns it toward the Gaseous Oxidizer Heat Exchanger (GOX HEX) coil. The cracking consistently initiated over a specific circumferential region of the hex vane, with other circumferential locations appearing with increased run time. Since cracking had not to date been seen with the baseline HPOTP, a fluid-structural interaction involving the ATD HPOTP turbine exit flowfield and the HEX inner vane was suspected. As part of NASA contract NAS8-36801, Pratt and Whitney conducted air flow tests of the ATD HPOTP turbine turnaround duct flowpath in the MSFC Phase 2 HGM air flow model. These tests included HEX vane strain gages and additional fluctuating pressure gages in the turnaround duct and HEX vane flowpath area. Three-dimensional flow probe measurements at two stations downstream of the turbine simulator exit plane were also made. Modifications to the HPOTP turbine simulator investigated the effects on turbine exit flow profile and velocity components, with the objective of reproducing flow conditions calculated for the actual ATD HPOTP hardware. Testing was done at the MSFC SSME Dynamic Fluid Air Flow (Dual-Leg) Facility, at air supply pressures between 50 and 250 psia. Combinations of turbine exit Mach number and pressure level were run to investigate the effect of flow regime. Information presented includes: (1) Descriptions of turbine simulator modifications to produce the desired flow environment; (2) Types and locations for instrumentation added to the flow model for improved diagnostic capability; (3) Evaluation of the effect of changes to the turbine simulator flowpath on the turbine exit flow environment; and (4

  13. Phase 2: HGM air flow tests in support of HEX vane investigation

    NASA Technical Reports Server (NTRS)

    Cox, G. B., Jr.; Steele, L. L.; Eisenhart, D. W.

    1993-01-01

    Following the start of SSME certification testing for the Pratt and Whitney Alternate Turbopump Development (ATD) High Pressure Oxidizer Turbopump (HPOTP), cracking of the leading edge of the inner HEX vane was experienced. The HEX vane, at the inlet of the oxidizer bowl in the Hot Gas Manifold (HGM), accepts the HPOTP turbine discharge flow and turns it toward the Gaseous Oxidizer Heat Exchanger (GOX HEX) coil. The cracking consistently initiated over a specific circumferential region of the hex vane, with other circumferential locations appearing with increased run time. Since cracking had not to date been seen with the baseline HPOTP, a fluid-structural interaction involving the ATD HPOTP turbine exit flowfield and the HEX inner vane was suspected. As part of NASA contract NAS8-36801, Pratt and Whitney conducted air flow tests of the ATD HPOTP turbine turnaround duct flowpath in the MSFC Phase 2 HGM air flow model. These tests included HEX vane strain gages and additional fluctuating pressure gages in the turnaround duct and HEX vane flowpath area. Three-dimensional flow probe measurements at two stations downstream of the turbine simulator exit plane were also made. Modifications to the HPOTP turbine simulator investigated the effects on turbine exit flow profile and velocity components, with the objective of reproducing flow conditions calculated for the actual ATD HPOTP hardware. Testing was done at the MSFC SSME Dynamic Fluid Air Flow (Dual-Leg) Facility, at air supply pressures between 50 and 250 psia. Combinations of turbine exit Mach number and pressure level were run to investigate the effect of flow regime. Information presented includes: (1) Descriptions of turbine simulator modifications to produce the desired flow environment; (2) Types and locations for instrumentation added to the flow model for improved diagnostic capability; (3) Evaluation of the effect of changes to the turbine simulator flowpath on the turbine exit flow environment; and (4

  14. Donor CD47 controls T cell alloresponses and is required for tolerance induction following hepatocyte allotransplantation

    PubMed Central

    Zhang, Mingyou; Wang, Hui; Tan, Shulian; Navarro-Alvarez, Nalu; Zheng, Yang; Yang, Yong-Guang

    2016-01-01

    CD47-deficient hepatocyte transplantation induces rapid innate immune cell activation and subsequent associated graft loss in syngeneic recipients. However, the role of donor CD47 in regulation of T-cell alloresponses is poorly understood. We addressed this question by assessing OVA-specific immune responses in mice following hepatocyte transplantation from CD47-competent or -deficient OVA-transgenic donors. Compared to sham-operated controls, intrasplenic transplantation of CD47-deficient OVA+ hepatocytes significantly accelerated rejection of OVA+ skin grafted 7 days after hepatocyte transplantation. In contrast, mice receiving CD47-competent OVA+ hepatocytes showed prolonged and even indefinite survival of OVA+ skin allografts. T cells from mice receiving CD47-deficient, but not CD47-competent, OVA+ hepatocytes showed significantly enhanced responses to OVA+ stimulators compared to sham-operated controls. In contrast to the production of tolerogenic cytokines (IL-4 and IL-10) in the recipients of CD47-competent hepatocytes, mice receiving CD47-deficient hepatocytes showed elevated production of IFN-γ and IL-1α. Moreover, significant expansion of myeloid-derived suppressor cells was detected in the recipients of CD47-competent hepatocytes, which was required for tolerance induction in these mice. Thus, donor CD47 plays an important role in the control of T-cell alloresponses and tolerance induction following hepatocyte transplantation. Our data also suggest that intrasplenic hepatocyte transplantation may provide a means to induce allograft tolerance. PMID:27230788

  15. Novel Mechanism of Impaired Function of Organic Anion-Transporting Polypeptide 1B3 in Human Hepatocytes: Post-Translational Regulation of OATP1B3 by Protein Kinase C Activation

    PubMed Central

    Powell, John; Farasyn, Taleah; Köck, Kathleen; Meng, Xiaojie; Pahwa, Sonia; Brouwer, Kim L. R.

    2014-01-01

    The organic anion-transporting polypeptide (OATP) 1B3 is a membrane transport protein that mediates hepatic uptake of many drugs and endogenous compounds. Currently, determination of OATP-mediated drug-drug interactions in vitro is focused primarily on direct substrate inhibition. Indirect inhibition of OATP1B3 activity is under-appreciated. OATP1B3 has putative protein kinase C (PKC) phosphorylation sites. Studies were designed to determine the effect of PKC activation on OATP1B3-mediated transport in human hepatocytes using cholecystokinin-8 (CCK-8), a specific OATP1B3 substrate, as the probe. A PKC activator, phorbol-12-myristate-13-acetate (PMA), did not directly inhibit [3H]CCK-8 accumulation in human sandwich-cultured hepatocytes (SCH). However, pretreatment with PMA for as little as 10 minutes rapidly decreased [3H]CCK-8 accumulation. Treatment with a PKC inhibitor bisindolylmaleimide (BIM) I prior to PMA treatment blocked the inhibitory effect of PMA, indicating PKC activation is essential for downregulating OATP1B3 activity. PMA pretreatment did not affect OATP1B3 mRNA or total protein levels. To determine the mechanism(s) underlying the indirect inhibition of OATP1B3 activity upon PKC activation, adenoviral vectors expressing FLAG-Myc-tagged OATP1B3 (Ad-OATP1B3) were transduced into human hepatocytes; surface expression and phosphorylation of OATP1B3 were determined by biotinylation and by an anti–phosphor-Ser/Thr/Tyr antibody, respectively. PMA pretreatment markedly increased OATP1B3 phosphorylation without affecting surface or total OATP1B3 protein levels. In conclusion, PKC activation rapidly decreases OATP1B3 transport activity by post-translational regulation of OATP1B3. These studies elucidate a novel indirect inhibitory mechanism affecting hepatic uptake mediated by OATP1B3, and provide new insights into predicting OATP-mediated drug interactions between OATP substrates and kinase modulator drugs/endogenous compounds. PMID:25200870

  16. The HEX 110 Hexamerin Is a Cytoplasmic and Nucleolar Protein in the Ovaries of Apis mellifera

    PubMed Central

    Martins, Juliana Ramos; Bitondi, Márcia Maria Gentile

    2016-01-01

    Hexamerins are insect storage proteins abundantly secreted by the larval fat body into the haemolymph. The canonical role of hexamerins consists of serving as an amino acid reserve for development toward the adult stage. However, in Apis mellifera, immunofluorescence assays coupled to confocal laser-scanning microscopy, and high-throughput sequencing, have recently shown the presence of hexamerins in other organs than the fat body. These findings have led us to study these proteins with the expectation of uncovering additional functions in insect development. We show here that a honeybee hexamerin, HEX 110, localizes in the cytoplasm and nucleus of ovarian cells. In the nucleus of somatic and germline cells, HEX 110 colocalized with a nucleolar protein, fibrillarin, suggesting a structural or even regulatory function in the nucleolus. RNase A provoked the loss of HEX 110 signals in the ovarioles, indicating that the subcellular localization depends on RNA. This was reinforced by incubating ovaries with pyronin Y, a RNA-specific dye. Together, the colocalization with fibrillarin and pyronin Y, and the sensitivity to RNase, highlight unprecedented roles for HEX110 in the nucleolus, the nuclear structure harbouring the gene cluster involved in ribosomal RNA production. However, the similar patterns of HEX 110 foci distribution in the active and inactive ovaries of queens and workers preclude its association with the functional status of these organs. PMID:26954256

  17. 5-Hydroxymethylfurfural protects against ER stress-induced apoptosis in GalN/TNF-α-injured L02 hepatocytes through regulating the PERK-eIF2α signaling pathway.

    PubMed

    Jiang, Ze-Qun; Ma, Yan-Xia; Li, Mu-Han; Zhan, Xiu-Qin; Zhang, Xu; Wang, Ming-Yan

    2015-12-01

    5-Hydroxymethylfurfural (5-HMF), a water-soluble compound extracted from wine-processed Fructus corni, is a novel hepatic protectant for treating acute liver injury. The present study was designed to investigate the protective effect of 5-HMF in human L02 hepatocytes injured by D-galactosamine (GalN) and tumor necrosis factor-α (TNF-α) in vitro and to explore the underlying mechanisms of action. Our results showed that 5-HMF caused significant increase in the viability of L02 cells injured by GalN/TNF-α, in accordance with a dose-dependent decrease in apoptotic cell death confirmed by morphological and flow cytometric analyses. Based on immunofluorescence and Western blot assays, we found that GalN/TNF-α induced ER stress in the cells, as indicated by the disturbance of intracellular Ca(2+) concentration, the activation of protein kinase RNA (PKR)-like ER kinase (PERK), phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α), and expression of ATF4 and CHOP proteins, which was reversed by 5-HMF pre-treatment in a dose-dependent manner. The anti-apoptotic effect of 5-HMF was further evidenced by balancing the expression of Bcl-2 family members. In addition, the knockdown of PERK suppressed the expression of phospho-PERK, phospho-eIF2α, ATF4, and CHOP, resulting in a significant decrease in cell apoptosis after the treatment with GalN/TNF-α. 5-HMF could enhance the effects of PERK knockdown, protecting the cells against the GalN/TNF-α insult. In conclusion, these findings demonstrate that 5-HMF can effectively protect GalN/TNF-α-injured L02 hepatocytes against ER stress-induced apoptosis through the regulation of the PERK-eIF2α signaling pathway, suggesting that it is a possible candidate for liver disease therapy. PMID:26721708

  18. Regulation of 3β-hydroxysteroid dehydrogenase and sulphotransferase 2A1 gene expression in primary porcine hepatocytes by selected sex-steroids and plant secondary metabolites from chicory (Cichorium intybus L.) and wormwood (Artemisia sp.).

    PubMed

    Rasmussen, Martin Krøyer; Ekstrand, Bo

    2014-02-15

    In pigs the endogenously produced compound androstenone is metabolised in the liver in two steps by 3β-hydroxysteroid dehydrogenase (3β-HSD) and sulphotransferase 2A1 (SULT2A1). The present study investigated the effect of selected sex-steroids (0.01-1 μM androstenone, testosterone and estradiol), skatole (1-100 μM) and secondary plant metabolites (1-100 μM) on the expression of 3β-HSD and SULT2A1 mRNA. Additionally the effect of a global methanolic extract of dried chicory root was investigated and compared to previous obtained in vivo effects. Primary hepatocytes were isolated from the livers of piglets (crossbreed: Landrace×Yorkshire and Duroc) and cultured for 24h before treatment for an additionally 24h. RNA was isolated from the hepatocytes and specific gene expression determined by RT-PCR using TaqMan probes. The investigated sex-steroids had no effect on the mRNA expression of 3β-HSD and SULT2A1, while skatole decreased the content of SULT2A1 30% compared to control. Of the investigated secondary plant metabolites artemisinin and scoparone (found in Artemisia sp.) lowered the content of SULT2A1 by 20 and 30% compared to control, respectively. Moreover, we tested three secondary plant metabolites (lactucin, esculetin and esculin) found in chicory root. Lactucin increased the mRNA content of both 3β-HSD and SULT2A1 by 200% compared to control. An extract of chicory root was shown to decrease the expression of both 3β-HSD and SULT2A1. It is concluded that the gene expression of enzymes with importance for androstenone metabolism is regulated by secondary plant metabolites in a complex manner. PMID:24333270

  19. Processing of kansui roots stir-baked with vinegar reduces kansui-induced hepatocyte cytotoxicity by decreasing the contents of toxic terpenoids and regulating the cell apoptosis pathway.

    PubMed

    Yan, Xiaojing; Zhang, Li; Guo, Jianming; Cao, Yudan; Shang, Erxin; Tang, Yuping; Ding, Anwei; Duan, Jin-Ao

    2014-01-01

    Euphorbia kansui is a Traditional Chinese Medicine widely used for the treatment of oedema, ascites and asthma. However, its serious hepatotoxicity hinders its safe clinical application. The process of stir-baking with vinegar is regularly used to reduce the toxicity of kansui. Up till now, the exact mechanism of the reduction in hepatotoxicity of kansui stir-baked with vinegar has been poorly defined. In this study, decreased  contents of five diterpene and one triterpene in kansui (GS-1) after stir-baking with vinegar (GS-2) was investigated by UPLC-QTOF/MS. Flow cytometry and Hoechst staining were used to show that the stir-baking with vinegar process reduces kansui-induced cell apoptosis. Furthermore, the result also indicated that kansui stir-baked with vinegar protects LO2 cells from apoptosis by increasing the cell mitochondrial membrane potential (ΔΨm), decreasing the release of cytochrome c and inhibiting the activities of caspase-9 and caspase-3 as evidenced by means of high content screening (HCS), ELISA and western blotting. These results suggested that the stir-baking vinegar could reduce the hepatotoxicity of kansui by effectively decreasing the contents of toxic terpenoids and inhibiting the intrinsic pathway of hepatocyte cell apoptosis. In conclusion, the study provided significant data for promoting safer and better clinical use of this herb. PMID:24896263

  20. Differential regulation of nitric oxide synthase mRNA expression by lipopolysaccharide and pro-inflammatory cytokines in fetal hepatocytes treated with cycloheximide.

    PubMed Central

    Casado, M; Díaz-Guerra, M J; Boscá, L; Martín-Sanz, P

    1997-01-01

    The effect of cycloheximide (CHX) on the mRNA expression of the cytokine-inducible, calcium-independent nitric oxide synthase (iNOS) was investigated in fetal hepatocytes stimulated with lipopolysaccharide (LPS) or pro-inflammatory cytokines. In the presence of CHX the LPS-dependent iNOS mRNA levels were reduced, whereas the response to pro-inflammatory cytokines was enhanced. Because iNOS transcription is highly dependent on the activation of nuclear factor kappaB (NF-kappaB), this factor was evaluated by electrophoretic mobility shift assays, and a close correlation between NF-kappaB activity and iNOS mRNA levels was observed. CHX itself potentiated the degradation of the IkappaB alpha and IkappaB beta inhibitory subunits (IkappaB is inhibitory kappaB) of the NF-kappaB complex, and therefore the loss of LPS-dependent iNOS mRNA expression cannot be attributed to a blockage in the activation of NF-kappaB. These results suggest the existence of a CHX-sensitive pathway in the expression of iNOS mediated by LPS, a mechanism that is not involved in the response to pro-inflammatory cytokines. PMID:9581561

  1. RNAi Screening in Primary Human Hepatocytes of Genes Implicated in Genome-Wide Association Studies for Roles in Type 2 Diabetes Identifies Roles for CAMK1D and CDKAL1, among Others, in Hepatic Glucose Regulation

    PubMed Central

    Haney, Steven; Zhao, Juan; Tiwari, Shiwani; Eng, Kurt; Guey, Lin T.; Tien, Eric

    2013-01-01

    Genome-wide association (GWA) studies have described a large number of new candidate genes that contribute to of Type 2 Diabetes (T2D). In some cases, small clusters of genes are implicated, rather than a single gene, and in all cases, the genetic contribution is not defined through the effects on a specific organ, such as the pancreas or liver. There is a significant need to develop and use human cell-based models to examine the effects these genes may have on glucose regulation. We describe the development of a primary human hepatocyte model that adjusts glucose disposition according to hormonal signals. This model was used to determine whether candidate genes identified in GWA studies regulate hepatic glucose disposition through siRNAs corresponding to the list of identified genes. We find that several genes affect the storage of glucose as glycogen (glycolytic response) and/or affect the utilization of pyruvate, the critical step in gluconeogenesis. Of the genes that affect both of these processes, CAMK1D, TSPAN8 and KIF11 affect the localization of a mediator of both gluconeogenesis and glycolysis regulation, CRTC2, to the nucleus in response to glucagon. In addition, the gene CDKAL1 was observed to affect glycogen storage, and molecular experiments using mutant forms of CDK5, a putative target of CDKAL1, in HepG2 cells show that this is mediated by coordinate regulation of CDK5 and PKA on MEK, which ultimately regulates the phosphorylation of ribosomal protein S6, a critical step in the insulin signaling pathway. PMID:23840313

  2. Three-Dimensional, Nodal, Neutron Diffusion Criticality Code System in Hex-Z Geometry.

    Energy Science and Technology Software Center (ESTSC)

    1992-07-27

    Version: 00 SIXTUS-3 is a 3D extention of SIXTUS-2 and is based on a response matrix nodal model. The code offers a fast and accurate analysis of critical systems in the regular hex-z geometry with the multigroup cross section representation including arbitrary upscattering.

  3. HexSim: A flexible simulation model for forecasting wildlife responses to multiple interacting stressors

    EPA Science Inventory

    With SERDP funding, we have improved upon a popular life history simulator (PATCH), and in doing so produced a powerful new forecasting tool (HexSim). PATCH, our starting point, was spatially explicit and individual-based, and was useful for evaluating a range of terrestrial lif...

  4. Fasting decreases apolipoprotein B mRNA editing and the secretion of small molecular weight apoB by rat hepatocytes: Evidence that the total amount of apoB secreted is regulated post-transcriptionally

    SciTech Connect

    Leighton, J.K.; Joyner, J.; Zamarripa, J.; Deines, M.; Davis, R.A. )

    1990-09-01

    Two different molecular weight forms of apoB are produced from a common initial transcript via editing of a Gln codon (CAA) to a stop codon (UAA), leading to a truncated translation product (apo BS) that consists of the amino terminal half of the larger form (apoBL). Previous studies have shown that fasting coordinately decreases lipogenesis and the secretion of very low density lipoprotein (VLDL) lipids and apoBS. Secretion of the apoBL is unaffected by fasting. We studied whether editing of apoB RNA is repressed by fasting, thus accounting for the selective decreased secretion of apoBS. Column chromatography of (35S)methionine-labeled lipoproteins secreted by hepatocytes from fed rats showed that essentially all of apoBL is secreted in the VLDL fraction, whereas a significant amount (15%) of apoBS is secreted associated as lipoproteins eluting in the HDL fractions. Fasting decreased the relative amount of apoBS that eluted in the VLDL fractions and increased the amount secreted in the HDL fractions. Consistent with previous results, hepatocytes from fasted rats show a selective twofold decrease in apoBS secretion. Fasting did not affect the relative abundance of apoB RNA, determined by slot blot hybridization assays using two different 32P-labeled cDNA probes coding either for both molecular weight forms or for only the large molecular weight form. However, quantitative of the editing of apoB RNA showed that fasting caused a 60% decrease in the amount of apoB RNA possessing the stop codon. These data show that the editing of apoB RNA is sensitive to metabolic state (i.e., fasting) resulting in a selective decrease in the secretion of apoBS. However, since the total secretion of apoB was decreased by fasting, while apoB mRNA levels remained constant, additional (post-transcriptional) mechanisms play a role in regulating apoB secretion.

  5. Hepatocyte-Ductal Transdifferentiation Is Mediated by Reciprocal Repression of SOX9 and C/EBPα

    PubMed Central

    O'Neill, Kathy E.; Thowfeequ, Shifaan; Li, Wan-Chun; Eberhard, Daniel; Dutton, James R.; Slack, Jonathan M.W.

    2014-01-01

    Abstract Primary hepatocytes rapidly dedifferentiate when cultured in vitro. We have studied the mechanism of hepatocyte dedifferentiation by using two culture media: one that maintains hepatocytes in a differentiated state and another that allows dedifferentiation. We show that dedifferentiation involves partial transformation of hepatocytes into cells that resemble biliary epithelial cells. Lineage labeling and time-lapse filming confirm that the dedifferentiated cells are derived from hepatocytes and not from contaminating ductal or fibroblastic cells in the original culture. Furthermore, we establish that the conversion of hepatocytes to biliary-like cells is regulated by mutual antagonism of CCAAT/enhancer binding protein alpha (C/EBPα) and SOX9, which have opposing effects on the expression of hepatocyte and ductal genes. Thus, hepatocyte dedifferentiation induces the biliary gene expression program by alleviating C/EBPα-mediated repression of Sox9. We propose that reciprocal antagonism of C/EBPα and SOX9 also operates in the formation of hepatocytes and biliary ducts from hepatoblasts during normal embryonic development. These data demonstrate that reprogramming of differentiated cells can be used to model the acquisition and maintenance of cell fate in vivo. PMID:25153359

  6. Death Receptor 5 Signaling Promotes Hepatocyte Lipoapoptosis*

    PubMed Central

    Cazanave, Sophie C.; Mott, Justin L.; Bronk, Steven F.; Werneburg, Nathan W.; Fingas, Christian D.; Meng, X. Wei; Finnberg, Niklas; El-Deiry, Wafik S.; Kaufmann, Scott H.; Gores, Gregory J.

    2011-01-01

    Nonalcoholic steatohepatitis is characterized by hepatic steatosis, elevated levels of circulating free fatty acids (FFA), endoplasmic reticulum (ER) stress, and hepatocyte lipoapoptosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor 5 (DR5) is significantly elevated in patients with nonalcoholic steatohepatitis, and steatotic hepatocytes demonstrate increased sensitivity to TRAIL-mediated cell death. Nonetheless, a role for TRAIL and/or DR5 in mediating lipoapoptotic pathways is unexplored. Here, we examined the contribution of DR5 death signaling to lipoapoptosis by free fatty acids. The toxic saturated free fatty acid palmitate induces an increase in DR5 mRNA and protein expression in Huh-7 human hepatoma cells leading to DR5 localization into lipid rafts, cell surface receptor clustering with subsequent recruitment of the initiator caspase-8, and ultimately cellular demise. Lipoapoptosis by palmitate was not inhibited by a soluble human recombinant DR5-Fc chimera protein suggesting that DR5 cytotoxic signaling is ligand-independent. Hepatocytes from murine TRAIL receptor knock-out mice (DR−/−) displayed reduced palmitate-mediated lipotoxicity. Likewise, knockdown of DR5 or caspase-8 expression by shRNA technology attenuated palmitate-induced Bax activation and apoptosis in Huh-7 cells, without altering induction of ER stress markers. Similar observations were verified in other cell models. Finally, knockdown of CHOP, an ER stress-mediated transcription factor, reduced DR5 up-regulation and DR5-mediated caspase-8 activation upon palmitate treatment. Collectively, these results suggest that ER stress-induced CHOP activation by palmitate transcriptionally up-regulates DR5, likely resulting in ligand-independent cytotoxic signaling by this death receptor. PMID:21941003

  7. Three Dimensional Primary Hepatocyte Culture

    NASA Technical Reports Server (NTRS)

    Yoffe, Boris

    1998-01-01

    Our results demonstrated for the first time the feasibility of culturing PHH in microgravity bioreactors that exceeded the longest period obtained using other methods. Within the first week of culture, isolated hepatocytes started to form aggregates, which continuously increased in size (up to 1 cm) and macroscopically appeared as a multidimensional tissue-like assembly. To improve oxygenation and nutrition within the spheroids we performed experiments with the biodegradable nonwoven fiber-based polymers made from PolyGlycolic Acid (PGA). It has been shown that PGA scaffolds stimulate isolated cells to regenerate tissue with defined sizes and shapes and are currently being studied for various tissue-engineering applications. Our data demonstrated that culturing hepatocytes in the presence of PGA scaffolds resulted in more efficient cell assembly and formations of larger cell spheroids (up to 3 cm in length, see figure). The histology of cell aggregates cultured with PGA showed polymer fibers with attached hepatocytes. We initiated experiments to co-culture primary human hepatocytes with human microvascular endothelial cells in the bioreactor. The presence of endothelial cells in co-cultures were established by immunohistochemistry using anti-CD34 monoclonal Ab. Our preliminary data demonstrated that cultures of purified hepatocytes with human microvascular endothelial cells exhibited better growth and expressed higher levels of albumin MRNA for a longer period of time than cultures of ppfified, primary human hepatocytes cultured alone. We also evaluated microsomal deethylation activity of hepatocytes cultured in the presence of endothelial cells.In summary, we have established liver cell culture, which mimicked the structure and function of the parent tissue.

  8. An Ingenol Derived from Euphorbia kansui Induces Hepatocyte Cytotoxicity by Triggering G0/G1 Cell Cycle Arrest and Regulating the Mitochondrial Apoptosis Pathway in Vitro.

    PubMed

    Yan, Xiaojing; Zhang, Li; Cao, Yudan; Yao, Weifeng; Tang, Yuping; Ding, Anwei

    2016-01-01

    Natural product lingenol, a purified diterpenoid compound derived from the root of Euphorbia kansui, exerts serious hepatotoxicity; however, the molecular mechanisms remain to be defined. In the present study, cell counting Kit-8 (CCK-8), inverted phase contrast microscope and flow cytometry were used to demonstrate that lingenol significantly inhibited L-O2 cells proliferation, and induced cell cycle arrest and apoptosis. Moreover, the results investigated that lingenol markedly disrupted mitochondrial functions by high content screening (HCS). In addition, the up-regulation of cytochrome c, AIF and Apaf-1 and activation of caspases were found in L-O2 cells detected by Western blotting and ELISA assay, which was required for lingenol activation of cytochrome c-mediated caspase cascades and AIF-mediated DNA damage. Mechanistic investigations revealed that lingenol significantly down-regulated the Bcl-2/Bax ratio and enhanced the reactive oxygen species (ROS) in L-O2 cells. These data collectively indicated that lingenol modulation of ROS and Bcl-2/Bax ratio led to cell cycle arrest and mitochondrial-mediated apoptosis in L-O2 cells in vitro. All of these results will be helpful to reveal the hepatotoxicity mechanism of Euphorbia kansui and to effectively guide safer and better clinical application of this herb. PMID:27338329

  9. Unsteady Flow Simulations in Support of the SSME HEX Turning Vane Cracking Investigation with the ATD HPOTP

    NASA Technical Reports Server (NTRS)

    Dougherty, N. S.; Burnette, D. W.; Holt, J. B.; Nesman, T.

    1993-01-01

    Unsteady flow computations are being performed with the P&W (ATD) and the Rocketdyne baseline configurations of the SSME LO2 turbine turnaround duct (TAD) and heat exchanger (HEX). The work is in support of the HEX inner turning vane cracking investigation. Fatigue cracking has occurred during hot firings with the P&W configuration on the HEX inner vane, and it appears the fix will involve changes to the TAD splitter vane position and to the TAD inner wall curvature to reduce the dynamic loading on the inner vane. Unsteady flow computations on the P&W baseline and fix and on the Rocketdyne baseline reference follow steady-flow screening computations done by MSFC/ED32 on several trial configurations arriving at the fix. The P&W TAD inlet velocity profile has a strong radial velocity component that directs the flow toward the inner wall and raises the local velocity a factor of two and the dynamic pressure a factor, of four. The fix is intended to redistribute the flow more evenly across the HEX inner and outer vanes like the Rocketdyne baseline reference. Vane buffeting at frequencies around 4,000 Hz is the leading suspected cause of the problem. Our simulations (work in progress) are being done with the USA 2D axisymmetric code approximating the flow as axisymmetric u+v 2D (axial, u, and radial, v, components only). The HEX coils are included in the model to make sure the fix does not adversely affect the HEX environment. Turbulent kinetic energy, k, levels where k = 1/2 v' rms2 are locally as high as 10,000 ft2/sec2 for the P&W baseline at the engine interface (between the TAD and HEX) at the HEX inner vane location. However, k is less than 8,000 on the HEX outer vane and only about 4,500 on the HEX inner vane for the Rocketdyne baseline. Unsteady turbulence intensity, v'rms/v, and pressure, p', are being computed in the present computations to compare with steady-flow Reynolds-averaged computations where p'rms = const (pk) for overall rms random turbulence from 0

  10. The xenobiotic-sensing nuclear receptors pregnane X receptor, constitutive androstane receptor, and orphan nuclear receptor hepatocyte nuclear factor 4alpha in the regulation of human steroid-/bile acid-sulfotransferase.

    PubMed

    Echchgadda, Ibtissam; Song, Chung S; Oh, Taesung; Ahmed, Mohamed; De La Cruz, Isidro John; Chatterjee, Bandana

    2007-09-01

    The nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are the primary transcription factors coordinating induced expression of the enzymes and proteins directing oxidative, conjugative, and transport phases of endobiotic and xenobiotic metabolism, whereas hepatocyte nuclear factor 4alpha (HNF4alpha), a regulator of hepatic lipid homeostasis, can modify the PXR/CAR response. Steroid- and bile acid-sulfotransferase (SULT2A1) promotes phase II metabolism through its sulfonating action on certain endobiotics, including steroids and bile acids, and on diverse xenobiotics, including therapeutic drugs. This study describes characterization of a PXR- and CAR-inducible composite element in the human SULT2A1 promoter and its synergistic interaction with HNF4alpha. Inverted and direct repeats of AG(G/T)TCA (IR2 and DR4), both binding to PXR and CAR, define the composite element. Differential recognition of the composite element by PXR and CAR is evident because single-site mutation at either IR2 or DR4 in the natural gene abolished the PXR response, whereas mutations at both repeats were necessary to abrogate completely the CAR response. The composite element conferred xenobiotic response to a heterologous promoter, and the cognate ligands induced PXR and CAR recruitment to the chromatin-associated response region. An HNF4alpha element adjacent to the -30 position enhanced basal promoter activity. Although functioning as a synergizer, the HNF4alpha element was not essential for the PXR/CAR response. An emerging role of SULT2A1 in lipid and caloric homeostasis suggests that illumination on the regulatory interactions driving human SULT2A1 expression may reveal new avenues to control certain metabolic disorders. PMID:17595319

  11. Kinetics and Dynamics of HEX to Gyroid Transition of a Diblock Copolymer in Selective Solvent

    NASA Astrophysics Data System (ADS)

    Spring, Julian; Liu, Yongsheng; Bansil, Rama

    2011-03-01

    Synchrotron based time-resolved small angle x-ray scattering (SAXS) was used to study the kinetics of the formation of a gyroid phase in solutions of a poly (styrene -isoprene) diblock copolymer in dimethyl phthalate, a selective solvent for the polystyrene block. From temperature ramp measurements on an 80% (w/v) sample, a hexagonally-packed cylinders (HEX) phase was identifed below 95 C,while a gyroid formed above 95C. The kinetics of the transitions from HEX to gyroid was examined using temperature jump and ramp experiments over the temperature range of 50-150C. In addition, x-ray photon correlation spectroscopy was used to study the dynamics of the HEX and Gyroid phases, as well as the transition regime. Analysis of the time evolution of the Bragg peaks to follow the kinetics of the transition between these phases will be presented, in addition to analysis of the dynamics of this sample throughout the phase space under investigation. The formation of the Gyroid structure was also modeled using Molecular Dynamics (MD) simulations, and the results of these simulations will also be presented.

  12. Characterization and expression of the Hex 110 gene encoding a glutamine-rich hexamerin in the honey bee, Apis mellifera.

    PubMed

    Bitondi, Márcia M G; Nascimento, Adriana M; Cunha, Adriana D; Guidugli, Karina R; Nunes, Francis M F; Simões, Zilá L P

    2006-10-01

    An N-terminal amino acid sequence of a previously reported honey bee hexamerin, HEX 110 [Danty et al., Insect Biochem Mol Biol 28:387-397 (1998)], was used as reference to identify the predicted genomic sequence in a public GenBank database. In silico analysis revealed an ORF of 3,033 nucleotides that encompasses eight exons. The conceptual translation product is a glutamine-rich polypeptide with a predicted molecular mass of 112.2 kDa and pI of 6.43, which contains the conserved M and C hemocyanin domains. Semiquantitative and quantitative RT-PCR with specific primers allowed for an analysis of mRNA levels during worker bee development and under different physiological conditions. Concomitantly, the abundance of the respective polypeptide in the hemolymph was examined by SDS-PAGE. Hex 110 transcripts were found in high levels during the larval stages, then decreased gradually during the pupal stage, and increased again in adults. HEX 110 subunits were highly abundant in larval hemolymph, decreased at the spinning-stage, and remained at low levels in pupae and adults. In 5th instar larvae, neither starvation nor supplementation of larval food with royal jelly changed the Hex 110 transcript levels or the amounts of HEX 110 subunit in hemolymph. In adult workers, high levels of Hex 110 mRNA, but not of the respective subunit, were related to ovary activation, and also to the consumption of a pollen-rich diet. PMID:16983665

  13. Hepatocyte cell therapy in liver disease.

    PubMed

    Bartlett, David Christopher; Newsome, Philip N

    2015-01-01

    Liver disease is a leading cause of morbidity and mortality. Liver transplantation remains the only proven treatment for end-stage liver failure but is limited by the availability of donor organs. Hepatocyte cell therapy, either with bioartificial liver devices or hepatocyte transplantation, may help address this by delaying or preventing liver transplantation. Early clinical studies have shown promising results, however in most cases, the benefit has been short lived and so further research into these therapies is required. Alternative sources of hepatocytes, including stem cell-derived hepatocytes, are being investigated as the isolation of primary human hepatocytes is limited by the same shortage of donor organs. This review summarises the current clinical experience of hepatocyte cell therapy together with an overview of possible alternative sources of hepatocytes. Current and future areas for research that might lead towards the realisation of the full potential of hepatocyte cell therapy are discussed. PMID:26212798

  14. Lipopolysaccharide Is Cleared from the Circulation by Hepatocytes via the Low Density Lipoprotein Receptor

    PubMed Central

    Topchiy, Elena; Cirstea, Mihai; Kong, HyeJin Julia; Boyd, John H.; Wang, Yingjin; Russell, James A.; Walley, Keith R.

    2016-01-01

    Sepsis is the leading cause of death in critically ill patients. While decreased Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) function improves clinical outcomes in murine and human sepsis, the mechanisms involved have not been fully elucidated. We tested the hypothesis that lipopolysaccharide (LPS), the major Gram-negative bacteria endotoxin, is cleared from the circulation by hepatocyte Low Density Lipoprotein Receptors (LDLR)—receptors downregulated by PCSK9. We directly visualized LPS uptake and found that LPS is rapidly taken up by hepatocytes into the cell periphery. Over the course of 4 hours LPS is transported towards the cell center. We next found that clearance of injected LPS from the blood was reduced substantially in Ldlr knockout (Ldlr-/-) mice compared to wild type controls and, simultaneously, hepatic uptake of LPS was also reduced in Ldlr-/- mice. Specifically examining the role of hepatocytes, we further found that primary hepatocytes isolated from Ldlr-/- mice had greatly decreased LPS uptake. In the HepG2 immortalized human hepatocyte cell line, LDLR silencing similarly resulted in decreased LPS uptake. PCSK9 treatment reduces LDLR density on hepatocytes and, therefore, was another independent strategy to test our hypothesis. Incubation with PCSK9 reduced LPS uptake by hepatocytes. Taken together, these findings demonstrate that hepatocytes clear LPS from the circulation via the LDLR and PCSK9 regulates LPS clearance from the circulation during sepsis by downregulation of hepatic LDLR. PMID:27171436

  15. Long non-coding RNAs expression profiles in hepatocytes of mice after hematopoietic stem cell transplantation.

    PubMed

    Qiao, Jianlin; Yao, Haina; Xia, Yuan; Chu, Peipei; Li, Mingfeng; Wu, Yulu; Li, Wen; Ding, Lan; Qi, Kunming; Li, Depeng; Xu, Kailin; Zeng, Lingyu

    2016-03-01

    Hepatic veno-occlusive disease (HVOD), one serious complication following hematopoietic stem cell transplantation (HSCT), is mainly initiated by the damage to sinusoidal endothelial cells and hepatocytes. Long non-coding RNAs (lncRNAs) play an important role in the proliferation of hepatocytes and liver regeneration. lncRNAs profile in hepatocytes post-HSCT remains unclear. The aim of this study is to evaluate the profile of lncRNAs in hepatocytes of mice after HSCT. Mice HSCT model was established through infusion of 5 × 10(6) bone marrow mononuclear cells. On day 7, 14 and 33 after HSCT, mice were sacrificed for analysis of liver pathology, function and index. Total RNA was extracted from hepatocytes of mice on day 14 for microarray analysis of the expression profiles of lncRNAs by Arraystar Mouse lncRNA Microarray v2.0. Obvious edema and spotty necrosis of hepatocytes with inflammatory cells infiltration were observed post-HSCT. Meanwhile, increased levels of alkaline phosphatase, aspartate transaminase, and total bilirubin, as well as elevated liver index were also found. 2,918 up-regulated and 1,911 down-regulated lncRNAs in hepatocytes were identified. Some of differentially expressed mRNAs had adjacent lncRNAs that were also significantly dysregulated, with the same dysregulation direction. T-cell receptor (up-regulation) and VEGF signaling pathway (down-regulation) were identified as one of the most enriched pathways. Dysregulated lncRNAs might be involved in hepatocytes damage after HSCT, suggesting targeting them might be a novel approach in amelioration of hepatocytes damage. PMID:26805554

  16. Hepatocytes: a key cell type for innate immunity.

    PubMed

    Zhou, Zhou; Xu, Ming-Jiang; Gao, Bin

    2016-05-01

    Hepatocytes, the major parenchymal cells in the liver, play pivotal roles in metabolism, detoxification, and protein synthesis. Hepatocytes also activate innate immunity against invading microorganisms by secreting innate immunity proteins. These proteins include bactericidal proteins that directly kill bacteria, opsonins that assist in the phagocytosis of foreign bacteria, iron-sequestering proteins that block iron uptake by bacteria, several soluble factors that regulate lipopolysaccharide signaling, and the coagulation factor fibrinogen that activates innate immunity. In this review, we summarize the wide variety of innate immunity proteins produced by hepatocytes and discuss liver-enriched transcription factors (e.g. hepatocyte nuclear factors and CCAAT/enhancer-binding proteins), pro-inflammatory mediators (e.g. interleukin (IL)-6, IL-22, IL-1β and tumor necrosis factor-α), and downstream signaling pathways (e.g. signal transducer and activator of transcription factor 3 and nuclear factor-κB) that regulate the expression of these innate immunity proteins. We also briefly discuss the dysregulation of these innate immunity proteins in chronic liver disease, which may contribute to an increased susceptibility to bacterial infection in patients with cirrhosis. PMID:26685902

  17. Hepatocytes: a key cell type for innate immunity

    PubMed Central

    Zhou, Zhou; Xu, Ming-Jiang; Gao, Bin

    2016-01-01

    Hepatocytes, the major parenchymal cells in the liver, play pivotal roles in metabolism, detoxification, and protein synthesis. Hepatocytes also activate innate immunity against invading microorganisms by secreting innate immunity proteins. These proteins include bactericidal proteins that directly kill bacteria, opsonins that assist in the phagocytosis of foreign bacteria, iron-sequestering proteins that block iron uptake by bacteria, several soluble factors that regulate lipopolysaccharide signaling, and the coagulation factor fibrinogen that activates innate immunity. In this review, we summarize the wide variety of innate immunity proteins produced by hepatocytes and discuss liver-enriched transcription factors (e.g. hepatocyte nuclear factors and CCAAT/enhancer-binding proteins), pro-inflammatory mediators (e.g. interleukin (IL)-6, IL-22, IL-1β and tumor necrosis factor-α), and downstream signaling pathways (e.g. signal transducer and activator of transcription factor 3 and nuclear factor-κB) that regulate the expression of these innate immunity proteins. We also briefly discuss the dysregulation of these innate immunity proteins in chronic liver disease, which may contribute to an increased susceptibility to bacterial infection in patients with cirrhosis. PMID:26685902

  18. Mechanisms of cortisol action in fish hepatocytes.

    PubMed

    Faught, Erin; Vijayan, Mathilakath M

    2016-09-01

    Here we provide an overview of the mechanistic characterization of the hepatic action of cortisol during stress in fish. Cortisol is the main circulating glucocorticoid in fish and its action is mediated through its cytosolic receptor, the glucocorticoid receptor (GR), and regulates the expression of genes involved in growth, metabolism and immune function. When taken together, the data suggests that cortisol may be playing a key role in the energy substrate re-partitioning in hepatocytes to cope with stress. The proposed model is that cortisol upregulates pathways involved in energy substrate mobilization, including gluconeogenesis, while downregulating energy demanding pathways, including growth and immune function. Recent work also points to a role for cortisol in mediating rapid action that is non-genomic and includes modulation of secondary signalling cascades; however, the physiological relevance of these studies remains to be determined. Altogether, studies carried out in hepatocytes are bringing to fore the complex nature of the cortisol signalling pathways in the organismal stress response. The mode of actions and their physiological implications for stress coping awaits further study. PMID:27445122

  19. Oncostatin M induces upregulation of claudin-2 in rodent hepatocytes coinciding with changes in morphology and function of tight junctions

    SciTech Connect

    Imamura, Masafumi; Kojima, Takashi . E-mail: ktakashi@sapmed.ac.jp; Lan, Mengdong; Son, Seiichi; Murata, Masaki; Osanai, Makoto; Chiba, Hideki; Hirata, Koichi; Sawada, Norimasa

    2007-05-15

    In rodent livers, integral tight junction (TJ) proteins claudin-1, -2, -3, -5 and -14 are detected and play crucial roles in the barrier to keep bile in bile canaculi away from the blood circulation. Claudin-2 shows a lobular gradient increasing from periportal to pericentral hepatocytes, whereas claudin-1 and -3 are expressed in the whole liver lobule. Although claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells, the physiological functions and regulation of claudin-2 in hepatocytes remain unclear. Oncostatin M (OSM) is a multifunctional cytokine implicated in the differentiation of hepatocytes that induces formation of E-cadherin-based adherens junctions in fetal hepatocytes. In this study, we examined whether OSM could induce expression and function of claudin-2 in rodent hepatocytes, immortalized mouse and primary cultured proliferative rat hepatocytes. In the immortalized mouse and primary cultured proliferative rat hepatocytes, treatment with OSM markedly increased mRNA and protein of claudin-2 together with formation of developed networks of TJ strands. The increase of claudin-2 enhanced the paracellular barrier function which depended on molecular size. The increase of claudin-2 expression induced by OSM in rodent hepatocytes was regulated through distinct signaling pathways including PKC. These results suggest that expression of claudin-2 in rodent hepatocytes may play a specific role as controlling the size of paracellular permeability in the barrier to keep bile in bile canaculi.

  20. Absorption coefficient, transition probability, and collision-broadening frequency of dimethylether at He-Xe laser 3.51-micron wavelength

    NASA Technical Reports Server (NTRS)

    Siegman, A. E.; Wang, S. C.

    1970-01-01

    Absorptivity, transition probability and collision broadening frequency of dimethylether at 3.51 micron He-Xe laser wavelength, noting pressure dependence, transition lifetime and saturation intensity

  1. Hypoxia-inducible Factor-dependent Production of Profibrotic Mediators by Hypoxic Hepatocytes

    PubMed Central

    Copple, Bryan L.; Bustamante, Juan J.; Welch, Timothy P.; Kim, Nam Deuk; Moon, Jeon-OK

    2011-01-01

    Background/Aims During the development of liver fibrosis, mediators are produced that stimulate cells in the liver to differentiate into myofibroblasts and to produce collagen. Recent studies demonstrated that the transcription factor, hypoxia-inducible factor-1α (HIF-1α), is critical for upregulation of profibrotic mediators, such as platelet-derived growth factor-A (PDGF-A), PDGF-B, and plasminogen activator inhibitor-1 (PAI-1) in the liver during the development of fibrosis. What remains unknown is the cell type-specific regulation of these genes by HIF-1α in liver cell types. Accordingly, the hypothesis was tested that HIF-1α is activated in hypoxic hepatocytes and regulates production of profibrotic mediators by these cells. Methods In this study, hepatocytes were isolated from the livers of control and HIF-1α or HIF-1β-Deficient mice and exposed to hypoxia. Results Exposure of primary mouse hepatocytes to 1% oxygen stimulated nuclear accumulation of HIF-1α and upregulated PAI-1, vascular endothelial cell growth factor, and the vasoactive peptides adrenomedullin-1 (ADM-1) and ADM-2. In contrast, levels of PDGF-A and PDGF-B mRNAs were unaffected in these cells by hypoxia. Exposure of HIF-1α-Deficient hepatocytes to 1% oxygen only partially prevented upregulation of these genes, suggesting that other hypoxia-regulated transcription factors, such as HIF-2α, may also regulate these genes. In support of this, HIF-2α was activated in hypoxic hepatocytes, and exposure of HIF-1β-Deficient hepatocytes to 1% oxygen completely prevented upregulation PAI-1, VEGF, and ADM-1, suggesting that HIF-2α may also contribute to upregulation of these genes in hypoxic hepatocytes. Conclusions Collectively, our results suggest that HIFs may be important regulators of profibrotic and vasoactive mediators by hypoxic hepatocytes. PMID:19302442

  2. Synthesis of anthracene derivatives of 1,3-diazabicyclo[3.1.0]hex-3-ene

    NASA Astrophysics Data System (ADS)

    Mahmoodi, Nosrat O.; Mirkhaef, Safoura; Ghavidast, Atefeh

    2015-02-01

    Novel mono- and bis-photochromic compounds of 1,3-diazabicyclo[3.1.0]hex-3-enes based on anthracene moiety were synthesized efficiently. Photochromic compounds were synthesized through the reaction of 10-(hydroxymethyl)anthracene-9-carbaldehyde and anthracene-9-carbaldehyde or 9,10-anthracenedicarbaldehyde as bis-aldehydes with ketoaziridines in dry DMF at room temperature. Photochromic compounds exhibited photochromic behavior both in solution and in solid state by irradiation under UV light at 254 nm. Compounds bearing 4-NO2 on aziridine moiety showed intensive color change. Compounds were characterized by IR, 1H NMR, 13C NMR, and UV-Vis.

  3. Synthesis of new pyrazolyl-1,3-diazabicyclo[3.1.0]hexe-3-ene derivatives

    NASA Astrophysics Data System (ADS)

    Kiyani, Hamzeh; Albooyeh, Fereshteh; Fallahnezhad, Saied

    2015-07-01

    A series of new of photochromic 1,3-diazabicyclo[3.1.0]hex-3-ene derivatives based on the skeleton of five-membered pyrazole moiety have been synthesized and characterized by spectral techniques, as well as their photochromic properties were examined under UV light irradiation in various solutions. All these newly synthesized compounds showed good photochromic properties in the both solution and solid states. The UV-Visible spectral analysis of the corresponding pyrazolyl bicyclic aziridines established structure-photochromic behavior relationships.

  4. Suppression of Autophagic Flux by Bile Acids in Hepatocytes

    PubMed Central

    Kong, Bo; Guo, Grace; Ding, Wen-Xing

    2014-01-01

    Retention of bile acids (BAs) in the liver during cholestasis plays an important role in the development of cholestatic liver injury. Several studies have reported that high concentrations of certain BAs induce cell death and inflammatory response in the liver, and BAs may promote liver tumorigenesis. Macroautophagy (hereafter referred to as autophagy) is a lysosomal degradation process that regulates organelle and protein homeostasis and serves as a cell survival mechanism under a variety of stress conditions. However, it is not known if BAs modulate autophagy in hepatocytes. In the present study, we determined autophagic flux in livers of farnesoid X receptor (FXR) knockout (KO) mice that have increased concentrations of hepatic BAs and in primary cultured mouse hepatocytes treated with BAs. The results showed that autophagic flux was impaired in livers of FXR KO mice and in BA-treated primary mouse hepatocytes. Mechanistically, BAs did not affect the activities of cathepsin or the proteasome, but impaired autophagosomal-lysosomal fusion likely due to reduction of Rab7 protein expression and targeting to autophagosomes. In conclusion, BAs suppress autophagic flux in hepatocytes by impairing autophagosomal-lysosomal fusion, which may be implicated in bile acid-induced liver tumor promotion observed in FXR KO mice. PMID:24189133

  5. Increased reprogramming of human fetal hepatocytes compared with adult hepatocytes in feeder-free conditions.

    PubMed

    Hansel, Marc C; Gramignoli, Roberto; Blake, William; Davila, Julio; Skvorak, Kristen; Dorko, Kenneth; Tahan, Veysel; Lee, Brian R; Tafaleng, Edgar; Guzman-Lepe, Jorge; Soto-Gutierrez, Alejandro; Fox, Ira J; Strom, Stephen C

    2014-01-01

    Hepatocyte transplantation has been used to treat liver disease. The availability of cells for these procedures is quite limited. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) may be a useful source of hepatocytes for basic research and transplantation if efficient and effective differentiation protocols were developed and problems with tumorigenicity could be overcome. Recent evidence suggests that the cell of origin may affect hiPSC differentiation. Thus, hiPSCs generated from hepatocytes may differentiate back to hepatocytes more efficiently than hiPSCs from other cell types. We examined the efficiency of reprogramming adult and fetal human hepatocytes. The present studies report the generation of 40 hiPSC lines from primary human hepatocytes under feeder-free conditions. Of these, 37 hiPSC lines were generated from fetal hepatocytes, 2 hiPSC lines from normal hepatocytes, and 1 hiPSC line from hepatocytes of a patient with Crigler-Najjar syndrome, type 1. All lines were confirmed reprogrammed and expressed markers of pluripotency by gene expression, flow cytometry, immunocytochemistry, and teratoma formation. Fetal hepatocytes were reprogrammed at a frequency over 50-fold higher than adult hepatocytes. Adult hepatocytes were only reprogrammed with six factors, while fetal hepatocytes could be reprogrammed with three (OCT4, SOX2, NANOG) or four factors (OCT4, SOX2, NANOG, LIN28 or OCT4, SOX2, KLF4, C-MYC). The increased reprogramming efficiency of fetal cells was not due to increased transduction efficiency or vector toxicity. These studies confirm that hiPSCs can be generated from adult and fetal hepatocytes including those with genetic diseases. Fetal hepatocytes reprogram much more efficiently than adult hepatocytes, although both could serve as useful sources of hiPSC-derived hepatocytes for basic research or transplantation. PMID:23394081

  6. Peripheral blood mononuclear cell infiltration and neuroinflammation in the HexB−/− mouse model of neurodegeneration

    PubMed Central

    Kyrkanides, Stephanos; Miller, Ann W.; Miller, Jen-nie H.; Tallents, Ross H.; Brouxhon, Sabine M.; Olschowka, Malory E.; O’Banion, M. Kerry; Olschowka, John A.

    2008-01-01

    Myeloid-derived immune cells, including microglia, macrophages and monocytes, have been previously implicated in neurodegeneration. We investigated the role of infiltrating peripheral blood mononuclear cells (PBMC) in neuroinflammation and neurodegeneration in the HexB−/− mouse model of Sandhoff disease. Ablation of the chemokine receptor CCR2 in the HexB−/− mouse resulted in significant inhibition of PBMC infiltration into the brain, decrease in TNFα and MHC-II mRNA abundance and retardation in clinical disease development. There was no change in the level of GM2 storage and pro-apoptotic activity or astrocyte activation in HexB−/−;Ccr2−/− double knockout mice, which eventually succumbed secondary to GM2 gangliosidosis. PMID:18657867

  7. Integration of Mesh Optimization with 3D All-Hex Mesh Generation, LDRD Subcase 3504340000, Final Report

    SciTech Connect

    KNUPP,PATRICK; MITCHELL,SCOTT A.

    1999-11-01

    In an attempt to automatically produce high-quality all-hex meshes, we investigated a mesh improvement strategy: given an initial poor-quality all-hex mesh, we iteratively changed the element connectivity, adding and deleting elements and nodes, and optimized the node positions. We found a set of hex reconnection primitives. We improved the optimization algorithms so they can untangle a negative-Jacobian mesh, even considering Jacobians on the boundary, and subsequently optimize the condition number of elements in an untangled mesh. However, even after applying both the primitives and optimization we were unable to produce high-quality meshes in certain regions. Our experiences suggest that many boundary configurations of quadrilaterals admit no hexahedral mesh with positive Jacobians, although we have no proof of this.

  8. Kinetics of Transition between HEX and Lamellar Phases in a triblock copolymer solution in a selective solvent.

    NASA Astrophysics Data System (ADS)

    Liu, Yongsheng; Bansil, Rama; Steinhart, Milos

    2006-03-01

    Synchrotron based time-resolved small angle x-ray scattering (SAXS), was used to study the kinetics of ordering transition (OOT) between cylindrical micelles in HEX phase and lamellar (LAM) phase in a 0.4 (w/v) solution of a triblock of polystyrene (PS) and poly(ethylene-co-butylene) (PEB), SEBS (PS-PEB-PS) copolymer in Dibutyl Phthalate (DBP), a selective solvent for the PS block. From a temperature ramp experiment the OOT was identified at about 137C and an ODT above 160 C. Several temperature jump experiments from HEX to LAM and the reverse were performed over the temperature range of 110-155C. Detailed analysis of the time evolution of the intensities of the Bragg peaks to follow the kinetics of the transition between HEX and LAM phases will be presented. A model to explain the transition mechanism will be discussed. This research was supported by NSF-DMR.

  9. Kinetics of Transition between HEX and Gyroid Phases in a Diblock Copolymer Solution in a Selective Solvent

    NASA Astrophysics Data System (ADS)

    Spring, Julian; Liu, Yongsheng; Bansil, Rama; Steinhart, Milos

    2010-03-01

    Synchrotron based time-resolved small angle x-ray scattering (SAXS), was used to study the kinetics of the formation of a gyroid phase in solutions of a poly (styrene - isoprene) diblock copolymer in dimethyl phthalate, a selective solvent for the polystyrene block. From temperature ramp measurements on a 75% (w/v) sample, a hexagonally-packed-cylinders (HEX) phase was identified below 110 C, a gyroid between 110 C and 150 C, above which the sample formed disordered spherical micelles. The kinetics of the transitions from HEX to gyroid, gyroid to disorder and disorder to gyroid was examined using temperature jump experiments over the temperature range of 5 165C. We found that the HEX to gyroid phase transition is irreversible, while gyroid to disorder is reversible. Detailed analysis of the time evolution of the Bragg peaks to follow the kinetics of the transition between these phases will be presented

  10. Blood-Compatible Polymer for Hepatocyte Culture with High Hepatocyte-Specific Functions toward Bioartificial Liver Development.

    PubMed

    Hoshiba, Takashi; Otaki, Takayuki; Nemoto, Eri; Maruyama, Hiroka; Tanaka, Masaru

    2015-08-19

    The development of bioartificial liver (BAL) is expected because of the shortage of donor liver for transplantation. The substrates for BAL require the following criteria: (a) blood compatibility, (b) hepatocyte adhesiveness, and (c) the ability to maintain hepatocyte-specific functions. Here, we examined blood-compatible poly(2-methoxyethyl acrylate) (PMEA) and poly(tetrahydrofurfuryl acrylate) (PTHFA) (PTHFA) as the substrates for BAL. HepG2, a human hepatocyte model, could adhere on PMEA and PTHFA substrates. The spreading of HepG2 cells was suppressed on PMEA substrates because integrin contribution to cell adhesion on PMEA substrate was low and integrin signaling was not sufficiently activated. Hepatocyte-specific gene expression in HepG2 cells increased on PMEA substrate, whereas the expression decreased on PTHFA substrates due to the nuclear localization of Yes-associated protein (YAP). These results indicate that blood-compatible PMEA is suitable for BAL substrate. Also, PMEA is expected to be used to regulate cell functions for blood-contacting tissue engineering. PMID:26258689

  11. Field experiments on high expansion (HEX) foam application for controlling LNG pool fire.

    PubMed

    Suardin, Jaffee A; Wang, Yanjun; Willson, Mike; Mannan, M Sam

    2009-06-15

    Previous research suggests that high expansion foam with an expansion ratio of 500 to 1 is one of the best options for controlling liquefied natural gas (LNG) pool fire on land. However, its effectiveness heavily depends on the foam application rate, foam generator location, and the design of LNG spill containment dike. Examination of these factors is necessary to achieve the maximum benefit for applying HEX on LNG pool fires. While theoretical study of the effects of foam on LNG fires is important, the complicated phenomena involved in LNG pool fire and foam application increase the need for LNG field experimentation. Therefore, five LNG experiments were conducted at Texas A&M University's Brayton Fire Training Field. ANGUS FIRE provided Expandol solution to form 500 to 1 high expansion foam (HEX) and its latest LNG Turbex Fixed High Expansion Foam Generators. In this paper, data collected during five experiments are presented and analyzed. The effectiveness of high expansion foam for controlling LNG pool fires with various application rates at two different types of containment pits is discussed. LNG fire behaviors and the effects of dike wall height are also presented and discussed. PMID:19056175

  12. Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis

    PubMed Central

    Cazanave, S C; Wang, X; Zhou, H; Rahmani, M; Grant, S; Durrant, D E; Klaassen, C D; Yamamoto, M; Sanyal, A J

    2014-01-01

    Non-alcoholic steatohepatitis is characterized by hepatic steatosis, elevated levels of circulating free fatty acids (FFA) and hepatocyte lipoapoptosis. This lipoapoptosis requires increased JNK phosphorylation and activation of the pro-apoptotic BH3-only proteins Bim and PUMA. Kelch-like ECH-associated protein (Keap)-1 is a BTB/Kelch protein that can regulate the expression of Bcl-2 protein and control apoptotic cell death. Yet, the role of Keap1 in hepatocyte lipotoxicity is unclear. Here we demonstrate that Keap1 protein was rapidly degraded in hepatocytes, through autophagy in a p62-dependent manner, in response to the toxic saturated FFA palmitate, but not following incubation with the non-toxic FFA oleic acid. Stable knockdown of Keap1 expression, using shRNA technology, in hepatocarcinoma cell lines induced spontaneous cell toxicity that was associated with JNK1-dependent upregulation of Bim and PUMA protein levels. Also, Keap1 knockdown further sensitized hepatocytes to lipoapoptosis by palmitate. Likewise, primary hepatocytes isolated from liver-specific Keap1−/− mice displayed higher Bim and PUMA protein levels and demonstrated increased sensitivity to palmitate-induced apoptosis than wild-type mouse hepatocytes. Finally, stable knockdown of Bim or PUMA expression prevented cell toxicity induced by loss of Keap1. These results implicate p62-dependent autophagic degradation of Keap1 by palmitate as a mechanism contributing to hepatocyte lipoapoptosis. PMID:24769730

  13. The immobilization of hepatocytes on 24 nm-sized gold colloid for enhanced hepatocytes proliferation.

    PubMed

    Gu, Hai-Ying; Chen, Zhong; Sa, Rong-Xiao; Yuan, Su-Su; Chen, Hong-Yuan; Ding, Yi-Tao; Yu, Ai-Min

    2004-08-01

    Bioartificial liver and hepatocyte transplantation is anticipated to supply a temporary metabolic support for candidates of liver transplantation or for patients with fulminant liver failure. An essential restriction of this form is the inability to acquire an enough amount of hepatocytes. Enhancement of the proliferation and differentiated function of hepatocytes is becoming a pursued target. Here, porcine hepatocytes were successfully immobilized on nano-sized gold colloid particles to construct a "hepatocyte/gold colloid" interface at which hepatocytes can be quickly proliferated. The properties of this resulting interface were characterized and confirmed by scanning electron microscopy and atomic force microscopy. The proliferative mechanism of hepatocytes was also discussed. The proliferated hepatocytes could be applied to the clinic based on their excellent functions for the synthesis of protein, glucose and urea as well as lower lactate dehydrogenase release. PMID:15020118

  14. Evidence that the sensitivity of carnitine palmitoyltransferase I to inhibition by malonyl-CoA is an important site of regulation of hepatic fatty acid oxidation in the fetal and newborn rabbit. Perinatal development and effects of pancreatic hormones in cultured rabbit hepatocytes.

    PubMed Central

    Prip-Buus, C; Pegorier, J P; Duee, P H; Kohl, C; Girard, J

    1990-01-01

    The temporal changes in oleate oxidation, lipogenesis, malonyl-CoA concentration and sensitivity of carnitine palmitoyltransferase I (CPT 1) to malonyl-CoA inhibition were studied in isolated rabbit hepatocytes and mitochondria as a function of time after birth of the animal or time in culture after exposure to glucagon, cyclic AMP or insulin. (1) Oleate oxidation was very low during the first 6 h after birth, whereas lipogenesis rate and malonyl-CoA concentration decreased rapidly during this period to reach levels as low as those found in 24-h-old newborns that show active oleate oxidation. (2) The changes in the activity of CPT I and the IC50 (concn. causing 50% inhibition) for malonyl-CoA paralleled those of oleate oxidation. (3) In cultured fetal hepatocytes, the addition of glucagon or cyclic AMP reproduced the changes that occur spontaneously after birth. A 12 h exposure to glucagon or cyclic AMP was sufficient to inhibit lipogenesis totally and to cause a decrease in malonyl-CoA concentration, but a 24 h exposure was required to induce oleate oxidation. (4) The induction of oleate oxidation by glucagon or cyclic AMP is triggered by the fall in the malonyl-CoA sensitivity of CPT I. (5) In cultured hepatocytes from 24 h-old newborns, the addition of insulin inhibits no more than 30% of the high oleate oxidation, whereas it stimulates lipogenesis and increases malonyl-CoA concentration by 4-fold more than in fetal cells (no oleate oxidation). This poor effect of insulin on oleate oxidation seems to be due to the inability of the hormone to increase the sensitivity of CPT I sufficiently. Altogether, these results suggest that the malonyl-CoA sensitivity of CPT I is the major site of regulation during the induction of fatty acid oxidation in the fetal rabbit liver. PMID:2167069

  15. Hypoxia remodels the composition of the constituent ceramide species of HexCer and Hex2Cer with phytosphingosine and hydroxy fatty acids in human colon cancer LS174T cells.

    PubMed

    Tanaka, Kouji; Tamiya-Koizumi, Keiko; Yamada, Masaki; Murate, Takashi; Kannagi, Reiji; Kyogashima, Mamoru

    2015-11-01

    Oxygen-requiring enzymes, such as Δ4-desaturase (dihydroceramide desaturase), sphingolipid Δ4-desaturase/C-4-hydroxylase, and fatty acid 2-hydroxylase are involved in ceramide synthesis. We prepared free ceramides, sphingomyelins and glycosphingolipids (GSLs) from cancer cells cultivated under conditions of normoxia and hypoxia, and analyzed these compounds using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Human colon cancer LS174T cells were employed because these cells highly express hydroxyl fatty acids and phytosphingosine (t18:0) which are expected to be greatly influenced by changes in oxygen levels. As expected, the populations of dihydro-species of free ceramide and sphingomyelin with C16:0 non-hydroxy fatty acid were elevated, and the populations of HexCers and Hex2Cers, composed of C16:0 or C16:0 hydroxy fatty acid (C16:0h), and sphingosine (d18:1) or t18:0, were decreased under hypoxia. However, appreciable populations of HexCer and Hex2Cer species of C24:0 or C24:0h and t18:0 remained. These results suggest that the individual species of GSLs with fatty acids possessing different alkyl chain lengths, either non-hydroxy fatty acids or hydroxyl fatty acids, may be metabolized individually. PMID:26194060

  16. Insights into transcriptional regulation of β-D-N-acetylhexosaminidase, an N-glycan-processing enzyme involved in ripening-associated fruit softening

    PubMed Central

    Irfan, Mohammad; Ghosh, Sumit; Kumar, Vinay; Chakraborty, Niranjan; Chakraborty, Subhra; Datta, Asis

    2014-01-01

    Tomato (Solanum lycopersicum) fruit ripening-specific N-glycan processing enzyme, β-D-N-acetylhexosaminidase (β-Hex), plays an important role in the ripening-associated fruit-softening process. However, the regulation of fruit ripening-specific expression of β-Hex is not well understood. We have identified and functionally characterized the fruit ripening-specific promoter of β-Hex and provided insights into its transcriptional regulation during fruit ripening. Our results demonstrate that RIPENING INHIBITOR (RIN), a global fruit ripening regulator, and ABSCISIC ACID STRESS RIPENING 1 (SlASR1), a poorly characterized ripening-related protein, are the transcriptional regulators of β-Hex. Both RIN and SlASR1 directly bound to the β-Hex promoter fragments containing CArG and C2-3(C/G)A cis-acting elements, the binding sites for RIN and SlASR1, respectively. Moreover, β-Hex expression/promoter activity in tomato fruits was downregulated once expression of either RIN or SlASR1 was suppressed; indicating that RIN and SlASR1 positively regulate the transcription of β-Hex during fruit ripening. Interestingly, RIN could also bind to the SlASR1 promoter, which contains several CArG cis-acting elements, and SlASR1 expression was suppressed in rin mutant fruits, indicating that RIN also acts as a positive regulator of SlASR1 expression during fruit ripening. Taken together, these results suggest that RIN, both directly and indirectly, through SlASR1, regulates the transcription of β-Hex during fruit ripening. The fruit ripening-specific promoter of β-Hex could be a useful tool in regulating gene expression during fruit ripening. PMID:25129131

  17. Differentiation of hepatocytes from pluripotent stem cells

    PubMed Central

    Mallanna, Sunil K.

    2014-01-01

    Differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells into hepatocyte-like cells provides a platform to study the molecular basis of human hepatocyte differentiation, to develop cell culture models of liver disease, and to potentially provide hepatocytes for treatment of end-stage liver disease. Additionally, hepatocyte-like cells generated from human pluripotent stem cells could serve as platforms for drug discovery, determination of pharmaceutical induced hepatotoxicity, and evaluation of idiosyncratic drug-drug interactions. Here, we describe a step-wise protocol previously developed in our laboratory that facilitates the highly efficient and reproducible differentiation of human pluripotent stem cells into hepatocyte-like cells. Our protocol uses defined culture conditions and closely recapitulates key developmental events that are found to occur during hepatogenesis. PMID:24510789

  18. Effect of abutment screw length and cyclic loading on removal torque in external and internal hex implants

    PubMed Central

    Mohammed, Hnd Hadi; Lee, Jin-Han; Bae, Ji-Myung

    2016-01-01

    PURPOSE The purpose of this study was to evaluate the effects of abutment screw length and cyclic loading on the removal torque (RTV) in external hex (EH) and internal hex (IH) implants. MATERIALS AND METHODS Forty screw-retained single crowns were connected to external and internal hex implants. The prepared titanium abutment screws were classified into 8 groups based on the number of threads (n = 5 per group): EH 12.5, 6.5, 3.5, 2.5 and IH 6.5, 5, 3.5, 2.5 threads. The abutment screws were tightened with 20 Ncm torque twice with 10-minute intervals. After 5 minutes, the initial RTVs of the abutment screws were measured with a digital torque gauge (MGT12). A customized jig was constructed to apply a load along the implant long axis at the central fossa of the maxillary first molar. The post-loading RTVs were measured after 16,000 cycles of mechanical loading with 50 N at a 1-Hz frequency. Statistical analysis included one-way analysis of variance and paired t-tests. RESULTS The post-loading RTVs were significantly lower than the initial RTVs in the EH 2.5 thread and IH 2.5 thread groups (P<.05). The initial RTVs exhibited no significant differences among the 8 groups, whereas the post-loading RTVs of the EH 6.5 and EH 3.5 thread groups were higher than those of the IH 3.5 thread group (P<.05). CONCLUSION Within the limitations of this study, the external hex implants with short screw lengths were more advantageous than internal hex implants with short screw lengths in torque maintenance after cyclic loading. PMID:26949489

  19. Developmental regulation of {beta}-hexosaminidase {alpha}- and {beta}-subunit gene expression in the rat reproductive system

    SciTech Connect

    Trasler, J.M.; Wakamatsu, N.; Gravel, R.A.; Benoit, G.

    1994-09-01

    {beta}-Hexosaminidase is an essential lysosomal enzyme whose absence in man results in a group of disorders, the G{sub M2} gangliosidoses. Enzyme activity for {beta}-hexosaminidase is many fold higher in the epididymis than in other tissues, is present in sperm and is postulated to be required for mammalian fertilization. To better understand how {beta}-hexosaminidase is regulated in the reproductive system, we quantitated the mRNA expression of the {alpha}- and {beta}-subunits (Hex {alpha} and Hex {beta}) of the enzyme in the developing rat testis and epididymis. Hex {alpha} mRNA was differentially expressed and abundant in adult rat testis and epididymis, 13- and 2-fold brain levels, respectively. In contrast, Hex {beta} mRNA levels in the testis and epididymis were .3- and 5-fold brain levels. Within the epididymis both Hex {alpha} and Hex {beta} mRNA concentrations were highest in the corpus, 1.5-fold and 9-fold initial segment values, respectively. During testis development from 7-91 days of age, testis levels of Hex {alpha} mRNA increased 10-fold and coincided with the appearance of spermatocytes and spermatids in the epithelium. In isolated male germ cells, Hex {alpha} expression was most abundant in haploid round spermatids. Hex {alpha} mRNA was undetectable after hypophysectomy and returned to normal after testosterone administration and the return of advanced germ cells to the testis. Hex {beta} mRNA was expressed at constant low levels throughout testis development. In the caput-corpus and cauda regions of the epididymis Hex {alpha} mRNA levels increased 2-fold between 14 and 91 days; during the same developmental period epididymal Hex {beta} mRNA levels increased dramatically, by 10-20 fold. In summary, Hex {alpha} and Hex {beta} mRNAs are differentially and developmentally expressed at high levels in the rat testis and epididymis and augur for an important role for {beta}-hexosaminidase in normal male reproductive function.

  20. LKB1/AMPK and PKA Control ABCB11 Trafficking and Polarization in Hepatocytes

    PubMed Central

    Homolya, László; Fu, Dong; Sengupta, Prabuddha; Jarnik, Michal; Gillet, Jean-Pierre; Vitale-Cross, Lynn; Gutkind, J. Silvio; Lippincott-Schwartz, Jennifer; Arias, Irwin M.

    2014-01-01

    Polarization of hepatocytes is manifested by bile canalicular network formation and activation of LKB1 and AMPK, which control cellular energy metabolism. The bile acid, taurocholate, also regulates development of the canalicular network through activation of AMPK. In the present study, we used collagen sandwich hepatocyte cultures from control and liver-specific LKB1 knockout mice to examine the role of LKB1 in trafficking of ABCB11, the canalicular bile acid transporter. In polarized hepatocytes, ABCB11 traffics from Golgi to the apical plasma membrane and endogenously cycles through the rab 11a-myosin Vb recycling endosomal system. LKB1 knockout mice were jaundiced, lost weight and manifested impaired bile canalicular formation and intracellular trafficking of ABCB11, and died within three weeks. Using live cell imaging, fluorescence recovery after photobleaching (FRAP), particle tracking, and biochemistry, we found that LKB1 activity is required for microtubule-dependent trafficking of ABCB11 to the canalicular membrane. In control hepatocytes, ABCB11 trafficking was accelerated by taurocholate and cAMP; however, in LKB1 knockout hepatocytes, ABCB11 trafficking to the apical membrane was greatly reduced and restored only by cAMP, but not taurocholate. cAMP acted through a PKA-mediated pathway which did not activate AMPK. Our studies establish a regulatory role for LKB1 in ABCB11 trafficking to the canalicular membrane, hepatocyte polarization, and canalicular network formation. PMID:24643070

  1. Growth inhibitory actions of prothrombin on normal hepatocytes: influence of matrix.

    PubMed

    Carr, Brian I; Kar, Siddhartha; Wang, Meifang; Wang, Ziqiu

    2007-09-01

    Most hepatomas have a defect in prothrombin carboxylation, and can secrete under-carboxylated prothrombin or des-gamma-carboxy-prothrombin (DCP), the function of which is unknown. We considered that the prothrombin-DCP axis might also be involved in growth control. Hepatocytes and hepatoma cells were treated with prothrombin and DNA synthesis and cytoskeletal changes were studied. Prothrombin inhibited DNA synthesis in hepatocytes on fibronectin, but not collagen matrix. Hepatoma cell lines were not inhibited. We found that hepatoma cell matrix conferred resistance to hepatocytes. Prothrombin decreased fibronectin but not collagen amounts, but only in the presence of hepatocytes and not hepatoma cells, indicating that it has a differential action on matrix proteins. It also caused changes in cell shape and actin depolymerization. In vivo, there was a decrease in plasma prothrombin activity after a partial hepatectomy (PH), concomitant with the peak of DNA synthesis in the hepatocytes at 24h after PH. Injection of warfarin at the time of PH, further inhibited PT activity and enhanced this 24h peak of DNA synthesis. Furthermore, repeated injection of prothrombin lowered the peak DNA synthesis after PH. The data support the hypothesis that prothrombin can act as a hepatocyte growth inhibitor, likely at the level of fibronectin loss and result in cytoskeletal changes. Hepatomas resist this action, possibly due to their different matrix proteins. This represents a novel mechanism for growth regulation and provides a possible biological significance for the tumor marker DCP. PMID:17490900

  2. LKB1/AMPK and PKA control ABCB11 trafficking and polarization in hepatocytes.

    PubMed

    Homolya, László; Fu, Dong; Sengupta, Prabuddha; Jarnik, Michal; Gillet, Jean-Pierre; Vitale-Cross, Lynn; Gutkind, J Silvio; Lippincott-Schwartz, Jennifer; Arias, Irwin M

    2014-01-01

    Polarization of hepatocytes is manifested by bile canalicular network formation and activation of LKB1 and AMPK, which control cellular energy metabolism. The bile acid, taurocholate, also regulates development of the canalicular network through activation of AMPK. In the present study, we used collagen sandwich hepatocyte cultures from control and liver-specific LKB1 knockout mice to examine the role of LKB1 in trafficking of ABCB11, the canalicular bile acid transporter. In polarized hepatocytes, ABCB11 traffics from Golgi to the apical plasma membrane and endogenously cycles through the rab 11a-myosin Vb recycling endosomal system. LKB1 knockout mice were jaundiced, lost weight and manifested impaired bile canalicular formation and intracellular trafficking of ABCB11, and died within three weeks. Using live cell imaging, fluorescence recovery after photobleaching (FRAP), particle tracking, and biochemistry, we found that LKB1 activity is required for microtubule-dependent trafficking of ABCB11 to the canalicular membrane. In control hepatocytes, ABCB11 trafficking was accelerated by taurocholate and cAMP; however, in LKB1 knockout hepatocytes, ABCB11 trafficking to the apical membrane was greatly reduced and restored only by cAMP, but not taurocholate. cAMP acted through a PKA-mediated pathway which did not activate AMPK. Our studies establish a regulatory role for LKB1 in ABCB11 trafficking to the canalicular membrane, hepatocyte polarization, and canalicular network formation. PMID:24643070

  3. A honey bee hexamerin, HEX 70a, is likely to play an intranuclear role in developing and mature ovarioles and testioles.

    PubMed

    Martins, Juliana R; Anhezini, Lucas; Dallacqua, Rodrigo P; Simões, Zilá L P; Bitondi, Márcia M G

    2011-01-01

    Insect hexamerins have long been known as storage proteins that are massively synthesized by the larval fat body and secreted into hemolymph. Following the larval-to-pupal molt, hexamerins are sequestered by the fat body via receptor-mediated endocytosis, broken up, and used as amino acid resources for metamorphosis. In the honey bee, the transcript and protein subunit of a hexamerin, HEX 70a, were also detected in ovaries and testes. Aiming to identify the subcellular localization of HEX 70a in the female and male gonads, we used a specific antibody in whole mount preparations of ovaries and testes for analysis by confocal laser-scanning microscopy. Intranuclear HEX 70a foci were evidenced in germ and somatic cells of ovarioles and testioles of pharate-adult workers and drones, suggesting a regulatory or structural role. Following injection of the thymidine analog EdU we observed co-labeling with HEX 70a in ovariole cell nuclei, inferring possible HEX 70a involvement in cell proliferation. Further support to this hypothesis came from an injection of anti-HEX 70a into newly ecdysed queen pupae where it had a negative effect on ovariole thickening. HEX 70a foci were also detected in ovarioles of egg laying queens, particularly in the nuclei of the highly polyploid nurse cells and in proliferating follicle cells. Additional roles for this storage protein are indicated by the detection of nuclear HEX 70a foci in post-meiotic spermatids and spermatozoa. Taken together, these results imply undescribed roles for HEX 70a in the developing gonads of the honey bee and raise the possibility that other hexamerins may also have tissue specific functions. PMID:22205988

  4. A Honey Bee Hexamerin, HEX 70a, Is Likely to Play an Intranuclear Role in Developing and Mature Ovarioles and Testioles

    PubMed Central

    Martins, Juliana R.; Anhezini, Lucas; Dallacqua, Rodrigo P.; Simões, Zilá L. P.; Bitondi, Márcia M. G.

    2011-01-01

    Insect hexamerins have long been known as storage proteins that are massively synthesized by the larval fat body and secreted into hemolymph. Following the larval-to-pupal molt, hexamerins are sequestered by the fat body via receptor-mediated endocytosis, broken up, and used as amino acid resources for metamorphosis. In the honey bee, the transcript and protein subunit of a hexamerin, HEX 70a, were also detected in ovaries and testes. Aiming to identify the subcellular localization of HEX 70a in the female and male gonads, we used a specific antibody in whole mount preparations of ovaries and testes for analysis by confocal laser-scanning microscopy. Intranuclear HEX 70a foci were evidenced in germ and somatic cells of ovarioles and testioles of pharate-adult workers and drones, suggesting a regulatory or structural role. Following injection of the thymidine analog EdU we observed co-labeling with HEX 70a in ovariole cell nuclei, inferring possible HEX 70a involvement in cell proliferation. Further support to this hypothesis came from an injection of anti-HEX 70a into newly ecdysed queen pupae where it had a negative effect on ovariole thickening. HEX 70a foci were also detected in ovarioles of egg laying queens, particularly in the nuclei of the highly polyploid nurse cells and in proliferating follicle cells. Additional roles for this storage protein are indicated by the detection of nuclear HEX 70a foci in post-meiotic spermatids and spermatozoa. Taken together, these results imply undescribed roles for HEX 70a in the developing gonads of the honey bee and raise the possibility that other hexamerins may also have tissue specific functions. PMID:22205988

  5. Highly Efficient Differentiation of Functional Hepatocytes From Human Induced Pluripotent Stem Cells

    PubMed Central

    Ma, Xiaocui; Tschudy-Seney, Benjamin; Roll, Garrett; Behbahan, Iman Saramipoor; Ahuja, Tijess P.; Tolstikov, Vladimir; Wang, Charles; McGee, Jeannine; Khoobyari, Shiva; Nolta, Jan A.; Willenbring, Holger

    2013-01-01

    Human induced pluripotent stem cells (hiPSCs) hold great potential for use in regenerative medicine, novel drug development, and disease progression/developmental studies. Here, we report highly efficient differentiation of hiPSCs toward a relatively homogeneous population of functional hepatocytes. hiPSC-derived hepatocytes (hiHs) not only showed a high expression of hepatocyte-specific proteins and liver-specific functions, but they also developed a functional biotransformation system including phase I and II metabolizing enzymes and phase III transporters. Nuclear receptors, which are critical for regulating the expression of metabolizing enzymes, were also expressed in hiHs. hiHs also responded to different compounds/inducers of cytochrome P450 as mature hepatocytes do. To follow up on this observation, we analyzed the drug metabolizing capacity of hiHs in real time using a novel ultraperformance liquid chromatography-tandem mass spectrometry. We found that, like freshly isolated primary human hepatocytes, the seven major metabolic pathways of the drug bufuralol were found in hiHs. In addition, transplanted hiHs engrafted, integrated, and proliferated in livers of an immune-deficient mouse model, and secreted human albumin, indicating that hiHs also function in vivo. In conclusion, we have generated a method for the efficient generation of hepatocytes from induced pluripotent stem cells in vitro and in vivo, and it appears that the cells function similarly to primary human hepatocytes, including developing a complete metabolic function. These results represent a significant step toward using patient/disease-specific hepatocytes for cell-based therapeutics as well as for pharmacology and toxicology studies. PMID:23681950

  6. ‘JD’ iPS cell–derived hepatocytes faithfully recapitulate the pathophysiology of familial hypercholesterolemia

    PubMed Central

    Cayo, Max A.; Cai, Jun; DeLaForest, Ann; Noto, Fallon K.; Nagaoka, Masato; Clark, Brian S.; Collery, Ross F.; Si-Tayeb, Karim; Duncan, Stephen A.

    2014-01-01

    Elevated levels of low density lipoprotein cholesterol (LDL-C) in plasma are a major contributor to cardiovascular disease (CVD), which is the leading cause of death worldwide. Genome–wide association studies (GWAS) have identified 95 loci that associate with control of lipid/cholesterol metabolism. Although GWAS results are highly provocative, direct analyses of the contribution of specific allelic variations in regulating LDL-C has been challenging due to the difficulty in accessing appropriate cells from affected patients. The primary cell type responsible for controlling cholesterol and lipid flux is the hepatocyte. Recently we have shown that cells with hepatocyte characteristics can be generated from human induced pluripotent stem cells (iPSC). This finding raises the possibility of using patient–specific iPSC–derived hepatocytes to study the functional contribution of GWAS loci in regulating lipid metabolism. To test the validity of this approach we produced iPSCs from a patient with mutations in the Low density lipoprotein receptor(LDLR) gene that result in familial hypercholesterolemia (FH). Conclusion: We demonstrate that 1) hepatocytes can be efficiently generated from FH iPSCs, 2) in contrast to control cells FH iPSC–derived hepatocytes are deficient in LDL–C uptake, 3) control but not FH iPS cell–derived hepatocytes increase LDL uptake in response to lovastatin, and 4) FH iPSC–derived hepatocytes display a marked elevation in secretion of lipidated ApoB-100. Cumulatively, these findings demonstrate that FH iPSC–derived hepatocytes recapitulate the complex pathophysiology of FH in culture. These results also establish that patient specific iPSC–derived hepatocytes could be used to definitively determine the functional contribution of allelic variation in regulating lipid and cholesterol metabolism and could potentially provide a platform for the identification of novel treatments of CVD. PMID:22653811

  7. Enzyme induction in cryopreserved human hepatocyte cultures.

    PubMed

    Kafert-Kasting, Sabine; Alexandrova, Krassimira; Barthold, Marc; Laube, Britta; Friedrich, Gerhard; Arseniev, Lubomir; Hengstler, Jan G

    2006-03-15

    Freshly isolated human hepatocytes are considered as the gold standard for in vitro testing of drug candidates. Meanwhile also cryopreserved human hepatocyte suspensions are available. However, a drawback of these cells is the incalculability of attachment to the culture dish. Therefore, we established a technique freezing hepatocytes cultured on a collagen gel. After thawing damaged cells were removed to a certain extent by gentle washing with culture medium prior to adding an upper gel layer. The morphology of the resulting hepatocyte cultures could not be distinguished from that of non-frozen cells. However, basal activities of cytochrome P450 isoforms decreased in cryopreserved compared to non-frozen hepatocytes, as evidenced by analysis of testosterone hydroxylation (OHT) in positions 6beta, 16alpha, 2beta and 6alpha. Nevertheless, enzyme induction factors caused by 24 h incubation with 50 microM rifampicin were similar in cryopreserved and non-frozen hepatocytes. In cryopreserved hepatocytes rifampicin caused an increase in mean values of 6beta-OHT formation from 57.2 to 157.7 pmol/well/min (2.8-fold), compared to an increase from 115.8 to 269.1 pmol/well/min (2.3-fold) in non-frozen cells. Similarly, 16alpha- and 2beta-OHT showed induction factors of 2.4- and 2.3-fold in cryopreserved compared to 1.6- and 2.4-fold in non-frozen hepatocytes, respectively. In conclusion, human hepatocytes cryopreserved on collagen gels show a clear induction of CYP3A4 by rifampicin, although the basal activities are reduced compared to non-frozen cells. PMID:16473453

  8. Hepatocyte growth factor-induced differentiation of bone mesenchymal stem cells toward hepatocyte-like cells occurs through nuclear factor-kappa B signaling in vitro.

    PubMed

    Yang, Tongxi; Wang, Yi; Jiang, Shasha; Liu, Xiaoping; Yu, Zhongjie

    2016-09-01

    Hepatocyte growth factor (HGF) is multifaceted cytokine that regulates proliferation, differentiation, morphology, and motility within numerous stem cells. More recently, HGF has been reported to induce the differentiation of bone mesenchymal stem cells (BMSCs) into mature hepatocytes, but the underlying biochemical and molecular signaling is largely unknown. We isolated BMSC from the bone marrow of rats, which were then cultured and exposed to HGF for 15 days. We subsequently assayed these cells for liver functionality and markers, and blocked NF-кB signaling at various stages of the pathway. The present results demonstrate that HGF induces the differentiation of BMSCs toward hepatocyte-like cells through the NF-кB signaling. More specifically, HGF upregulated the translocation of NF-кB to the nucleus. PMID:27249785

  9. Differential Impacts of Soybean and Fish Oils on Hepatocyte Lipid Droplet Accumulation and Endoplasmic Reticulum Stress in Primary Rabbit Hepatocytes

    PubMed Central

    Zhu, Xueping; Xiao, Zhihui; Xu, Yumin; Zhao, Xingli; Cheng, Ping; Cui, Ningxun; Cui, Mingling; Li, Jie; Zhu, Xiaoli

    2016-01-01

    Parenteral nutrition-associated liver disease (PNALD) is a severe ailment associated with long-term parenteral nutrition. Soybean oil-based lipid emulsions (SOLE) are thought to promote PNALD development, whereas fish oil-based lipid emulsions (FOLE) are thought to protect against PNALD. This study aimed to investigate the effects of SOLE and FOLE on primary rabbit hepatocytes. The results reveal that SOLE caused significant endoplasmic reticulum (ER) and mitochondrial damage, ultimately resulting in lipid droplets accumulation and ER stress. While these deleterious events induce hepatocyte injury, FOLE at high doses cause only minor ER and mitochondrial damage, which has no effect on hepatic function. SOLE also significantly upregulated glucose-regulated protein 94 mRNA and protein expression. These data indicate that SOLE, but not FOLE, damage the ER and mitochondria, resulting in lipid droplets accumulation and ER stress and, finally, hepatocyte injury. This likely contributes to the differential impacts of SOLE and FOLE on PNALD development and progression. PMID:27057162

  10. Aging lowers steady-state antioxidant enzyme and stress protein expression in primary hepatocytes.

    PubMed

    Hall, D M; Sattler, G L; Sattler, C A; Zhang, H J; Oberley, L W; Pitot, H C; Kregel, K C

    2001-06-01

    It has been reported that the isolation and culture of primary hepatocytes can compromise cellular ability to constituitively express antioxidant enzyme (AE) genes, making it difficult to study their regulation ex vivo. In the present study, the steady-state expression of manganese-containing superoxide dismutase, copper- and zinc-containing superoxide dismutase, catalase, and glutathione peroxidase was assessed in primary hepatocytes isolated from young and senescent rats and cultured in MATRIGEL: There was no change in steady-state superoxide dismutase protein or activity levels in cells collected from young animals and cultured for 7 days. Catalase expression was initially increased, and then it declined 30%. In contrast, superoxide dismutase expression declined 60% and catalase expression declined 50% in cells from senescent animals. Constitutive and inducible 70-kDa heat shock protein expression increased coincident with declining AE levels in the young cells but not senescent cells. For both age groups, electron micrographs showed rounded hepatocytes with abundant rough endoplasmic reticulum, mitochondria, and peroxisomes. Hepatocytes were organized into clusters of 6-12 cells surrounding a large central lumen devoid of microvilli. Each cluster also contained smaller microvilli-lined lumens between adjacent hepatocytes that resembled canniculi. The plasma membranes of these lumens were sealed from the extracellular space by junctional complexes. Gap junctions in the plasma membrane suggest that hepatocytes were capable of intercellular communication. We conclude that the Matrigel system can be used to study AE regulation in primary hepatocytes from young and senescent animals, provided that experiments can be conducted within a time frame of 5-7 days in culture. These data also support the hypothesis that aging compromises hepatocellular ability to maintain AE status and upregulate stress protein expression. PMID:11382788

  11. SIRT1 Disruption in Human Fetal Hepatocytes Leads to Increased Accumulation of Glucose and Lipids

    PubMed Central

    Tobita, Takamasa; Guzman-Lepe, Jorge; Takeishi, Kazuki; Nakao, Toshimasa; Wang, Yang; Meng, Fanying; Deng, Chu-Xia; Collin de l’Hortet, Alexandra; Soto-Gutierrez, Alejandro

    2016-01-01

    There are unprecedented epidemics of obesity, such as type II diabetes and non-alcoholic fatty liver diseases (NAFLD) in developed countries. A concerning percentage of American children are being affected by obesity and NAFLD. Studies have suggested that the maternal environment in utero might play a role in the development of these diseases later in life. In this study, we documented that inhibiting SIRT1 signaling in human fetal hepatocytes rapidly led to an increase in intracellular glucose and lipids levels. More importantly, both de novo lipogenesis and gluconeogenesis related genes were upregulated upon SIRT1 inhibition. The AKT/FOXO1 pathway, a major negative regulator of gluconeogenesis, was decreased in the human fetal hepatocytes inhibited for SIRT1, consistent with the higher level of gluconeogenesis. These results indicate that SIRT1 is an important regulator of lipid and carbohydrate metabolisms within human fetal hepatocytes, acting as an adaptive transcriptional response to environmental changes. PMID:26890260

  12. SIRT1 Disruption in Human Fetal Hepatocytes Leads to Increased Accumulation of Glucose and Lipids.

    PubMed

    Tobita, Takamasa; Guzman-Lepe, Jorge; Takeishi, Kazuki; Nakao, Toshimasa; Wang, Yang; Meng, Fanying; Deng, Chu-Xia; Collin de l'Hortet, Alexandra; Soto-Gutierrez, Alejandro

    2016-01-01

    There are unprecedented epidemics of obesity, such as type II diabetes and non-alcoholic fatty liver diseases (NAFLD) in developed countries. A concerning percentage of American children are being affected by obesity and NAFLD. Studies have suggested that the maternal environment in utero might play a role in the development of these diseases later in life. In this study, we documented that inhibiting SIRT1 signaling in human fetal hepatocytes rapidly led to an increase in intracellular glucose and lipids levels. More importantly, both de novo lipogenesis and gluconeogenesis related genes were upregulated upon SIRT1 inhibition. The AKT/FOXO1 pathway, a major negative regulator of gluconeogenesis, was decreased in the human fetal hepatocytes inhibited for SIRT1, consistent with the higher level of gluconeogenesis. These results indicate that SIRT1 is an important regulator of lipid and carbohydrate metabolisms within human fetal hepatocytes, acting as an adaptive transcriptional response to environmental changes. PMID:26890260

  13. Hepatic stellate cell-expressed endosialin balances fibrogenesis and hepatocyte proliferation during liver damage.

    PubMed

    Mogler, Carolin; Wieland, Matthias; König, Courtney; Hu, Junhao; Runge, Anja; Korn, Claudia; Besemfelder, Eva; Breitkopf-Heinlein, Katja; Komljenovic, Dorde; Dooley, Steven; Schirmacher, Peter; Longerich, Thomas; Augustin, Hellmut G

    2015-03-01

    Liver fibrosis is a reversible wound-healing response to injury reflecting the critical balance between liver repair and scar formation. Chronic damage leads to progressive substitution of liver parenchyma by scar tissue and ultimately results in liver cirrhosis. Stromal cells (hepatic stellate cells [HSC] and endothelial cells) have been proposed to control the balance between liver fibrosis and regeneration. Here, we show that endosialin, a C-type lectin, expressed in the liver exclusively by HSC and portal fibroblasts, is upregulated in liver fibrosis in mouse and man. Chronic chemically induced liver damage resulted in reduced fibrosis and enhanced hepatocyte proliferation in endosialin-deficient (EN(KO)) mice. Correspondingly, acute-liver-damage-induced hepatocyte proliferation (partial hepatectomy) was increased in EN(KO) mice. A candidate-based screen of known regulators of hepatocyte proliferation identified insulin-like growth factor 2 (IGF2) as selectively endosialin-dependent hepatocyte mitogen. Collectively, the study establishes a critical role of HSC in the reciprocal regulation of fibrogenesis vs. hepatocyte proliferation and identifies endosialin as a therapeutic target in non-neoplastic settings. PMID:25680861

  14. Hepatic stellate cell-expressed endosialin balances fibrogenesis and hepatocyte proliferation during liver damage

    PubMed Central

    Mogler, Carolin; Wieland, Matthias; König, Courtney; Hu, Junhao; Runge, Anja; Korn, Claudia; Besemfelder, Eva; Breitkopf-Heinlein, Katja; Komljenovic, Dorde; Dooley, Steven; Schirmacher, Peter; Longerich, Thomas; Augustin, Hellmut G

    2015-01-01

    Liver fibrosis is a reversible wound-healing response to injury reflecting the critical balance between liver repair and scar formation. Chronic damage leads to progressive substitution of liver parenchyma by scar tissue and ultimately results in liver cirrhosis. Stromal cells (hepatic stellate cells [HSC] and endothelial cells) have been proposed to control the balance between liver fibrosis and regeneration. Here, we show that endosialin, a C-type lectin, expressed in the liver exclusively by HSC and portal fibroblasts, is upregulated in liver fibrosis in mouse and man. Chronic chemically induced liver damage resulted in reduced fibrosis and enhanced hepatocyte proliferation in endosialin-deficient (ENKO) mice. Correspondingly, acute-liver-damage-induced hepatocyte proliferation (partial hepatectomy) was increased in ENKO mice. A candidate-based screen of known regulators of hepatocyte proliferation identified insulin-like growth factor 2 (IGF2) as selectively endosialin-dependent hepatocyte mitogen. Collectively, the study establishes a critical role of HSC in the reciprocal regulation of fibrogenesis vs. hepatocyte proliferation and identifies endosialin as a therapeutic target in non-neoplastic settings. PMID:25680861

  15. Fibroblast growth factor 7 inhibits cholesterol 7{alpha}-hydroxylase gene expression in hepatocytes

    SciTech Connect

    Sun, Zhichao; Yu, Xuemei; Wu, Weibin; Jia, Dongwei; Chen, Yinle; Ji, Lingling; Liu, Xijun; Peng, Xiaomin; Li, Yintao; Yang, Lili; Ruan, Yuanyuan; Gu, Jianxin; Ren, Shifang; Zhang, Songwen

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer FGF7 strongly and rapidly down-regulates the expression of CYP7A1 in hepatocytes. Black-Right-Pointing-Pointer FGF7 suppresses the expression of CYP7A1 via FGFR2 and downstream JNK activation. Black-Right-Pointing-Pointer Blocking FGF7 abrogates HSC-induced inhibition of CYP7A1 expression in hepatocytes. -- Abstract: Cholesterol 7{alpha}-hydroxylase (CYP7A1) is the initial and rate-limiting enzyme for bile acid synthesis. Transcription of the CYP7A1 gene is regulated by bile acids, nuclear receptors and cytokines. Fibroblast growth factor 7 (FGF7) secreted from activated hepatic stellate cells (HSC) during chronic liver fibrosis regulates hepatocyte survival and liver regeneration. In the carbon tetrachloride (CCl{sub 4})-induced fibrotic mouse liver, we demonstrated that the expression of CYP7A1 was largely decreased while the expression of FGF7 was significantly increased. We further demonstrated that FGF7 inhibited CYP7A1 gene expression in hepatocytes. Knockdown study by short interfering RNA, kinase inhibition and phosphorylation assays revealed that the suppression of CYP7A1 expression by FGF7 was mediated by FGFR2 and its downstream JNK signaling cascade. The FGF7 neutralizing antibody restored CYP7A1 expression in Hep3B cells treated with conditioned medium from HSC. In summary, the data suggest that FGF7 is a novel regulator of CYP7A1 expression in hepatocytes and may prevent hepatocytes from accumulating toxic bile acids during liver injury and fibrosis.

  16. Use of mRNA expression to detect the induction of drug metabolising enzymes in rat and human hepatocytes

    SciTech Connect

    Richert, L. Tuschl, G.; Pekthong, D.; Mantion, G.; Weber, J.-C.; Mueller, S.O.

    2009-02-15

    It is important to investigate the induction of cytochrome P450 (CYP) enzymes by drugs. The most relevant end point is enzyme activity; however, this requires many cells and is low throughput. We have compared the CYP1A, CYP2B and CYP3A induction response to eight inducers in rat and human hepatocytes using enzyme activities (CYP1A2 (ethoxyresorufin), 2B (benzoxyresorufin for rat and bupropion for human) and CYP3A (testosterone)) and Taqman{sup TM} Low Density Array (TLDA) analysis. There was a good correlation between the induction of CYP1A2, CYP2B6 and CYP3A4 enzyme activities and mRNA expression in human hepatocytes. In contrast, BROD activities and mRNA expression in rat hepatocytes correlated poorly. However, bupropion hydroxylation correlated well with Cyp2b1 expression in rat hepatocytes. TLDA analysis of a panel of mRNAs encoding for CYPs, phase 2 enzymes, nuclear receptors and transporters revealed that the main genes induced by the 8 compounds tested were the CYPs. AhR ligands also induced UDP-glucuronosyltransferases and glutathione S-transferases in rat and human hepatocytes. The transporters, MDR1, MDR3 and OATPA were the only transporter genes significantly up-regulated in human hepatocytes. In rat hepatocytes Bsep, Mdr2, Mrp2, Mrp3 and Oatp2 were up-regulated. We could then show a good in vivo:in vitro correlation in the induction response of isolated rat hepatocytes and ex-vivo hepatic microsomes for the drug development candidate, EMD392949. In conclusion, application of TLDA methodology to investigate the potential of compounds to induce enzymes in rat and human hepatocytes increases the throughput and information gained from one assay, without reducing the predictive capacity.

  17. Molecular analysis of beta-hexosaminidase A (Hex A) deficiency among persons of French Canadian background living in New England

    SciTech Connect

    Triggs-Raine, B.; Richard, M.; Wasel, N.

    1994-09-01

    Tay-Sachs disease (TSD) results from mutations in the HEXA gene that cause Hex A deficiency. Enzyme screening for disease prevention has been applied in the Ashkenazi Jewish and French Canadian populations which have an elevated disease incidence. However, benign mutations that cause Hex A deficiency, but not TSD, complicate enzyme screening programs. While benign mutations account for only about 2% of Jewish carriers, they account for about 36% of non-Jewish enzyme-defined carriers. We have found a carrier frequency of 1/72 (n=1300) among persons of French Canadian background living in New England using an enzyme-based assay. The HEXA gene of these carriers and others was analyzed to determine the molecular basis of Hex A deficiency in this group. DNA samples were tested for common previously identified mutations; samples in which no change was found were screened for uncommon or novel mutations using SSCP analysis. Exons showing mobility shifts were sequenced and most mutations were confirmed by restriction enzyme digestion. Known disease-causing mutations were found in 8 samples (4 had a 7.6 kb deletion found in 80% of French Canadian TSD alleles) and known benign mutations were found in 4 samples. Seven novel mutations (G748A; +18 IVS-10 G-to-A; T1338C; +94 IVS-14 T-to-G; C1164G; +30 IVS-6 T-to-G) were identified; the G748A (Gly250Ser) change was found in 3 samples. The effects of the novel mutations on Hex A is unknown; some are likely polymorphisms. The molecular basis of this carrier population is clearly different from that of French Canadian TSD patients. Screening centers should be aware of the presence of benign mutations in the French Canadian population. Given the frequency of the Gly250Ser mutation, and the fact that it has been detected in a TSD patient, it too may be benign.

  18. Genes for the dimerization cofactor of hepatocyte nuclear factor-1[alpha] (DCOH) are on human and murine chromsomes 10

    SciTech Connect

    Milatovich, A.; Mendel, D.B.; Crabtree, G.R.; Francke, U. )

    1993-04-01

    Hepatocyte nuclear factor-1[alpha] (HNF-1[alpha]; gene symbol, TCF1) forms dimers with itself as well as with HNF-1[beta] and regulates the expression of several liver-specific genes. Recently, a dimerization cofactor of hepatocyte nuclear factor-1[alpha], called DCOH, has been identified. Here, the authors report the chromosomal localization of the genes for this cofactor to chromosomes 10 in both humans and mice by Southern blot analyses of somatic cell hybrids. 25 refs., 1 fig., 2 tabs.

  19. Activation of factor X by rat hepatocytes

    SciTech Connect

    Willingham, A.K.; Matschiner, J.T.

    1986-05-01

    Synthesis and secretion of blood coagulation factor X was studied in hepatocytes prepared by perfusion of rat livers with collagenase. Hepatocytes were incubated in the presence of vitamin K and /sup 3/H-leucine for up to 4h at 37/sup 0/C. Factor X was isolated from the incubation medium by immunochemical techniques and analyzed by SDS-PAGE. The recovered /sup 3/H-labeled proteins migrated, after reduction of disulfides, as two polypeptide chains with apparent molecular weights (M/sub r/) of approximately 42,000 and 22,000 representing the heavy and light chains of factor X respectively. The apparent M/sub r/ of the heavy chain was about 10,000 daltons lighter than seen with the heavy chain of factor X isolated from rat plasma and was more characteristic of the heavy chain of factor Xa. When the levels of factor X secreted by hepatocytes were determined by clotting assays, activity was present as factor Xa. Also, when purified plasma factor X was added to incubations of hepatocytes (>95% parenchymal cells) the added factor X was rapidly converted to factor Xa. Plasma membranes prepared from isolated hepatocytes or from liver homogenates contained an enzyme that converted factor X to factor Xa in a calcium dependent reaction. The physiological significance of a factor X activating enzyme on hepatocyte plasma membranes is not clear.

  20. Selective insulin resistance in hepatocyte senescence

    SciTech Connect

    Aravinthan, Aloysious; Challis, Benjamin; Shannon, Nicholas; Hoare, Matthew; Heaney, Judith; Alexander, Graeme J.M.

    2015-02-01

    Insulin resistance has been described in association with chronic liver disease for decades. Hepatocyte senescence has been demonstrated in chronic liver disease and as many as 80% of hepatocytes show a senescent phenotype in advanced liver disease. The aim of this study was to understand the role of hepatocyte senescence in the development of insulin resistance. Senescence was induced in HepG2 cells via oxidative stress. The insulin metabolic pathway was studied in control and senescent cells following insulin stimulation. GLUT2 and GLUT4 expressions were studied in HepG2 cells and human liver tissue. Further, GLUT2 and GLUT4 expressions were studied in three independent chronic liver disease cohorts. Signalling impairment distal to Akt in phosphorylation of AS160 and FoxO1 was evident in senescent HepG2 cells. Persistent nuclear localisation of FoxO1 was demonstrated in senescent cells despite insulin stimulation. Increased GLUT4 and decreased GLUT2 expressions were evident in senescent cells, human cirrhotic liver tissue and publically available liver disease datasets. Changes in GLUT expressions were associated with a poor clinical prognosis. In conclusion, selective insulin resistance is evident in senescent HepG2 cells and changes in GLUT expressions can be used as surrogate markers of hepatocyte senescence. - Highlights: • Senescent hepatocytes demonstrate selective insulin resistance. • GLUT changes act as markers of hepatocyte senescence and have prognostic value. • Study offers insight into long noticed intimacy of cirrhosis and insulin resistance.

  1. Development of a HEX-Z Partially Homogenized Benchmark Model for the FFTF Isothermal Physics Measurements

    SciTech Connect

    John D. Bess

    2012-05-01

    A series of isothermal physics measurements were performed as part of an acceptance testing program for the Fast Flux Test Facility (FFTF). A HEX-Z partially-homogenized benchmark model of the FFTF fully-loaded core configuration was developed for evaluation of these measurements. Evaluated measurements include the critical eigenvalue of the fully-loaded core, two neutron spectra, 32 reactivity effects measurements, an isothermal temperature coefficient, and low-energy gamma and electron spectra. Dominant uncertainties in the critical configuration include the placement of radial shielding around the core, reactor core assembly pitch, composition of the stainless steel components, plutonium content in the fuel pellets, and boron content in the absorber pellets. Calculations of criticality, reactivity effects measurements, and the isothermal temperature coefficient using MCNP5 and ENDF/B-VII.0 cross sections with the benchmark model are in good agreement with the benchmark experiment measurements. There is only some correlation between calculated and measured spectral measurements; homogenization of many of the core components may have impacted computational assessment of these measurements. This benchmark evaluation has been added to the IRPhEP Handbook.

  2. Radiographic analysis of hexapod external fixators: fundamental differences between the Taylor Spatial Frame and TrueLok-Hex.

    PubMed

    Ferreira, Nando; Birkholtz, Franz

    2015-04-01

    Hexapod circular external fixators are extremely accurate at correcting deformities in three dimensions. In order to exploit this accuracy, however, the surgeon must be able to accurately analyse the deformity and mounting parameters on post-operative radiographs. A Sawbone® model was created to simulate a mid-shaft tibial fracture with deformity. A 180 mm ring was applied oblique to the proximal segment of the Sawbone® model, in both the sagittal and coronal planes. Standard radiographs were taken of the model and analysed using the described Taylor Spatial Frame and TrueLok-Hex methods. The TrueLok-Hex software allows the surgeon the ability to program reference rings that are not orthogonally mounted. Apart from this software difference, the described analysis methods resulted in variation in all translational measurements for both deformity and mounting parameters. In conclusion, the radiographic analysis of the Taylor Spatial Frame and TrueLok-Hex are fundamentally different. These differences must be appreciated in order to use these systems effectively. PMID:25786500

  3. The HEX1 gene of Fusarium graminearum is required for fungal asexual reproduction and pathogenesis and for efficient viral RNA accumulation of Fusarium graminearum virus 1.

    PubMed

    Son, Moonil; Lee, Kyung-Mi; Yu, Jisuk; Kang, Minji; Park, Jin Man; Kwon, Sun-Jung; Kim, Kook-Hyung

    2013-09-01

    The accumulation of viral RNA depends on many host cellular factors. The hexagonal peroxisome (Hex1) protein is a fungal protein that is highly expressed when the DK21 strain of Fusarium graminearum virus 1 (FgV1) infects its host, and Hex1 affects the accumulation of FgV1 RNA. The Hex1 protein is the major constituent of the Woronin body (WB), which is a peroxisome-derived electron-dense core organelle that seals the septal pore in response to hyphal wounding. To clarify the role of Hex1 and the WB in the relationship between FgV1 and Fusarium graminearum, we generated targeted gene deletion and overexpression mutants. Although neither HEX1 gene deletion nor overexpression substantially affected vegetative growth, both changes reduced the production of asexual spores and reduced virulence on wheat spikelets in the absence of FgV1 infection. However, the vegetative growth of deletion and overexpression mutants was increased and decreased, respectively, upon FgV1 infection compared to that of an FgV1-infected wild-type isolate. Viral RNA accumulation was significantly decreased in deletion mutants but was significantly increased in overexpression mutants compared to the viral RNA accumulation in the virus-infected wild-type control. Overall, these data indicate that the HEX1 gene plays a direct role in the asexual reproduction and virulence of F. graminearum and facilitates viral RNA accumulation in the FgV1-infected host fungus. PMID:23864619

  4. Exosomes derived from alcohol-treated hepatocytes horizontally transfer liver specific miRNA-122 and sensitize monocytes to LPS

    PubMed Central

    Momen-Heravi, Fatemeh; Bala, Shashi; Kodys, Karen; Szabo, Gyongyi

    2015-01-01

    Hepatocyte damage and inflammation in monocytes/macrophages are central to the pathogenesis of alcoholic hepatitis (AH). MicroRNAs (miRNAs) regulate all of these processes. MiRNA-122 is abundantly expressed in hepatocytes while monocytes/macrophages have low levels. The role of exosomes in AH and possible cross talk between hepatocyte-derived exosomes and immune cells is not explored yet. Here, we show that the number of exosomes significantly increases in the sera of healthy individuals after alcohol binge drinking and in mice after binge or chronic alcohol consumption. Exosomes isolated from sera after alcohol consumption or from in vitro ethanol-treated hepatocytes contained miRNA-122. Exosomes derived from ethanol-treated Huh7.5 cells were taken up by the recipients THP1 monocytes and horizontally transferred a mature form of liver-specific miRNA-122. In vivo, liver mononuclear cells and Kupffer cells from alcohol-fed mice had increased miRNA-122 levels. In monocytes, miRNA-122 transferred via exosomes inhibited the HO-1 pathway and sensitized to LPS stimulation and increased levels of pro-inflammatory cytokines. Finally, inflammatory effects of exosomes from ethanol-treated hepatocytes were prevented by using RNA interference via exosome-mediated delivery of a miRNA-122 inhibitor. These results demonstrate that first, exosomes mediate communication between hepatocytes and monocytes/macrophages and second, hepatocyte-derived miRNA-122 can reprogram monocytes inducing sensitization to LPS. PMID:25973575

  5. Exosomes derived from alcohol-treated hepatocytes horizontally transfer liver specific miRNA-122 and sensitize monocytes to LPS.

    PubMed

    Momen-Heravi, Fatemeh; Bala, Shashi; Kodys, Karen; Szabo, Gyongyi

    2015-01-01

    Hepatocyte damage and inflammation in monocytes/macrophages are central to the pathogenesis of alcoholic hepatitis (AH). MicroRNAs (miRNAs) regulate all of these processes. MiRNA-122 is abundantly expressed in hepatocytes while monocytes/macrophages have low levels. The role of exosomes in AH and possible cross talk between hepatocyte-derived exosomes and immune cells is not explored yet. Here, we show that the number of exosomes significantly increases in the sera of healthy individuals after alcohol binge drinking and in mice after binge or chronic alcohol consumption. Exosomes isolated from sera after alcohol consumption or from in vitro ethanol-treated hepatocytes contained miRNA-122. Exosomes derived from ethanol-treated Huh7.5 cells were taken up by the recipients THP1 monocytes and horizontally transferred a mature form of liver-specific miRNA-122. In vivo, liver mononuclear cells and Kupffer cells from alcohol-fed mice had increased miRNA-122 levels. In monocytes, miRNA-122 transferred via exosomes inhibited the HO-1 pathway and sensitized to LPS stimulation and increased levels of pro-inflammatory cytokines. Finally, inflammatory effects of exosomes from ethanol-treated hepatocytes were prevented by using RNA interference via exosome-mediated delivery of a miRNA-122 inhibitor. These results demonstrate that first, exosomes mediate communication between hepatocytes and monocytes/macrophages and second, hepatocyte-derived miRNA-122 can reprogram monocytes inducing sensitization to LPS. PMID:25973575

  6. Nature and mechanisms of hepatocyte apoptosis induced by d-galactosamine/lipopolysaccharide challenge in mice

    PubMed Central

    WU, YI-HANG; HU, SHAO-QING; LIU, JUN; CAO, HONG-CUI; XU, WEI; LI, YONG-JUN; LI, LAN-JUAN

    2014-01-01

    Apoptosis plays a role in the normal development of liver. However, overactivation thereof may lead to hepatocellular damage. The aim of this study was to assess d-galactosamine (d-GalN)/lipopolysaccharide (LPS)-induced hepatocyte apoptotic changes in mice and clarify the mechanisms involved in this process. DNA ladder detection was employed to determine the induction condition of hepatic apoptosis. An initial test indicated that typical hepatocyte apoptosis was observed at 6–10 h after the intraperitoneal injection of d-GalN (700 mg/kg) and LPS (10 μg/kg). Subsequently, we evaluated hepatocyte apoptosis at 8 h after administering d-GalN/LPS by histopathological analysis, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) detection, flow cytometry and electron microscopy analysis. To clarify the apoptosis-related gene expression, the expression levels of tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), caspase-3, and Fas/Fas ligand (FasL) were determined by serum enzyme immunoassay, immunohistochemistry and western blot analysis. Strong apoptotic positive signals following d-GalN/LPS injection were observed from the results of the serum analysis, histopathological and immunohistochemical analyses, DNA ladder detection, TUNEL detection, flow cytometry and electron microscopy analysis. Additionally, apoptotic hepatocytes were mainly at the late stage of cell apoptosis. The expression of TNF-α, TGF-β1, caspase-3 and Fas/FasL was significantly increased. In conclusion, this study evaluated the d-GalN/LPS-induced hepatocyte apoptotic changes and clarified the apoptosis-related gene expression in mice. The hepatocyte apoptosis induced by d-GalN/LPS may be mainly regulated by the death receptor pathway. TGF-β signaling pathway may also play a vital role in this process of hepatocyte apoptosis. PMID:24714963

  7. Applicability of second-generation upcyte® human hepatocytes for use in CYP inhibition and induction studies

    PubMed Central

    Ramachandran, Sarada D; Vivarès, Aurélie; Klieber, Sylvie; Hewitt, Nicola J; Muenst, Bernhard; Heinz, Stefan; Walles, Heike; Braspenning, Joris

    2015-01-01

    Human upcyte® hepatocytes are proliferating hepatocytes that retain many characteristics of primary human hepatocytes. We conducted a comprehensive evaluation of the application of second-generation upcyte® hepatocytes from four donors for inhibition and induction assays using a selection of reference inhibitors and inducers. CYP1A2, CYP2B6, CYP2C9, and CYP3A4 were reproducibly inhibited in a concentration-dependent manner and the calculated IC50 values for each compound correctly classified them as potent inhibitors. Upcyte® hepatocytes were responsive to prototypical CYP1A2, CYP2B6, CYP2C9, and CYP3A4 inducers, confirming that they have functional AhR-, CAR-, and PXR-mediated CYP regulation. A panel of 11 inducers classified as potent, moderate or noninducers of CYP3A4 and CYP2B6 were tested. There was a good fit of data from upcyte® hepatocytes to three different predictive models for CYP3A4 induction, namely the Relative Induction Score (RIS), AUCu/F2, and Cmax,u/Ind50. In addition, PXR (rifampicin) and CAR-selective (carbamazepine and phenytoin) inducers of CYP3A4 and CYP2B6 induction, respectively, were demonstrated. In conclusion, these data support the use of second-generation upcyte® hepatocytes for CYP inhibition and induction assays. Under the culture conditions used, these cells expressed CYP activities that were equivalent to or higher than those measured in primary human hepatocyte cultures, which could be inhibited or induced by prototypical CYP inhibitors and inducers, respectively. Moreover, they can be used to predict in vivo CYP3A4 induction potential using three prediction models. Bulk availability of cells from multiple donors makes upcyte® hepatocytes suitable for DDI screening, as well as more in-depth mechanistic investigations. PMID:26516577

  8. Resistin impairs SIRT1 function and induces senescence-associated phenotype in hepatocytes.

    PubMed

    Yu, An; Zheng, Yu; Zhang, Rongrong; Huang, Jianfeng; Zhu, Zhoujie; Zhou, Ronghua; Jin, Dan; Yang, Zaiqing

    2013-09-01

    Resistin is a cysteine-rich secreted protein which significantly inhibits phosphorylation of AMP-activated protein kinase in both human and mouse hepatocytes. It has been demonstrated that resistin plays an important role in inducing hepatic insulin resistance. However, whether resistin has other unknown influences on hepatocytes still remains poorly studied. Here, we show that recombinant resistin protein significantly reduces expression of SIRT1, attenuates the interaction between SIRT1 and PPARα as well as PGC-1α, and increases PGC-1α acetyl-lysine levels in HepG2 cells. In line with this, resistin treatment weakens the association between SIRT1 and major satellite repeats and alters the transcription level of SIRT1 target genes in mouse primary hepatocytes. Resistin treatment also significantly increases senescence-associated β-galactosidase activity in mouse primary hepatocytes and this effect can be eliminated by co-treatment with the SIRT1 agonists resveratrol and nicotinamide mononucleotide. Our findings suggest that resistin is a negative regulator of SIRT1 in both human hepatoma cell line HepG2 and mouse hepatocytes and that it might also play an important role in the development of senescence-associated liver diseases. PMID:23827175

  9. Role of a liver fatty acid-binding protein gene in lipid metabolism in chicken hepatocytes.

    PubMed

    Gao, G L; Na, W; Wang, Y X; Zhang, H F; Li, H; Wang, Q G

    2015-01-01

    This study investigated the role of the chicken liver fatty acid-binding protein (L-FABP) gene in lipid metabolism in hepatocytes, and the regulatory relationships between L-FABP and genes related to lipid metabolism. The short hairpin RNA (shRNA) interference vector with L-FABP and an eukaryotic expression vector were used. Chicken hepatocytes were subjected to shRNA-mediated knockdown or L-FABP cDNA overexpression. Expression levels of lipid metabolism-related genes and biochemical parameters were detected 24, 36, 48, 60, and 72 h after transfection with the interference or overexpression plasmids for L-FABP, PPARα and L-BABP expression levels, and the total amount of cholesterol, were significantly affected by L-FABP expression. L-FABP may affect lipid metabolism by regulating PPARα and L-BABP in chicken hepatocytes. PMID:25966259

  10. Induction of hepatocyte lipopolysaccharide binding protein in models of sepsis and the acute-phase response.

    PubMed

    Geller, D A; Kispert, P H; Su, G L; Wang, S C; Di Silvio, M; Tweardy, D J; Billiar, T R; Simmons, R L

    1993-01-01

    Lipopolysaccharide binding protein (LBP) is a serum glycoprotein that complexes with lipopolysaccharide (LPS) to facilitate macrophage response to endotoxin. To determine the conditions that stimulate LBP production in vivo, we measured the induction of LBP in models of inflammation produced by LPS, Corynebacterium parvum, and turpentine injection. Plasma aspartate aminotransferase and alanine aminotransferase concentrations and hepatocyte fibrinogen synthesis were elevated in all models. Northern blot analysis revealed 17-, 14-, and 20-fold upregulation of hepatocyte LBP mRNA following treatment with LPS, C parvum, and turpentine, respectively. Peritoneal macrophage interleukin 6 and tumor necrosis factor production following endotoxin stimulation was augmented by cultured hepatocyte supernatants, suggesting increased LBP synthesis in these groups. The results show that LBP mRNA is induced during hepatic inflammation and suggest that LBP is an acute-phase protein important in regulating the in vivo response to endotoxin. PMID:8418776

  11. Macrophage Migration Inhibitor Factor Upregulates MCP-1 Expression in an Autocrine Manner in Hepatocytes during Acute Mouse Liver Injury.

    PubMed

    Xie, Jieshi; Yang, Le; Tian, Lei; Li, Weiyang; Yang, Lin; Li, Liying

    2016-01-01

    Macrophage migration inhibitor factor (MIF), a multipotent innate immune mediator, is an upstream component of the inflammatory cascade in diseases such as liver disease. Monocyte chemoattractant protein-1 (MCP-1), a highly representative chemokine, is critical in liver disease pathogenesis. We investigated the role of MIF in regulating hepatocytic MCP-1 expression. MIF and MCP-1 expression were characterized by immunochemistry, RT-PCR, ELISA, and immunoblotting in CCl4-treated mouse liver and isolated hepatocytes. MIF was primarily distributed in hepatocytes, and its expression increased upon acute liver injury. Its expression was also increased in injured hepatocytes, induced by LPS or CCl4, which mimic liver injury in vitro. MIF was expressed earlier than MCP-1, strongly inducing hepatocytic MCP-1 expression. Moreover, the increase in MCP-1 expression induced by MIF was inhibited by CD74- or CD44-specific siRNAs and SB203580, a p38 MAPK inhibitor. Further, CD74 or CD44 deficiency effectively inhibited MIF-induced p38 activation. MIF inhibitor ISO-1 reduced MCP-1 expression and p38 phosphorylation in CCl4-treated mouse liver. Our results showed that MIF regulates MCP-1 expression in hepatocytes of injured liver via CD74, CD44, and p38 MAPK in an autocrine manner, providing compelling information on the role of MIF in liver injury, and implying a new regulatory mechanism for liver inflammation. PMID:27273604

  12. Macrophage Migration Inhibitor Factor Upregulates MCP-1 Expression in an Autocrine Manner in Hepatocytes during Acute Mouse Liver Injury

    PubMed Central

    Xie, Jieshi; Yang, Le; Tian, Lei; Li, Weiyang; Yang, Lin; Li, Liying

    2016-01-01

    Macrophage migration inhibitor factor (MIF), a multipotent innate immune mediator, is an upstream component of the inflammatory cascade in diseases such as liver disease. Monocyte chemoattractant protein-1 (MCP-1), a highly representative chemokine, is critical in liver disease pathogenesis. We investigated the role of MIF in regulating hepatocytic MCP-1 expression. MIF and MCP-1 expression were characterized by immunochemistry, RT-PCR, ELISA, and immunoblotting in CCl4-treated mouse liver and isolated hepatocytes. MIF was primarily distributed in hepatocytes, and its expression increased upon acute liver injury. Its expression was also increased in injured hepatocytes, induced by LPS or CCl4, which mimic liver injury in vitro. MIF was expressed earlier than MCP-1, strongly inducing hepatocytic MCP-1 expression. Moreover, the increase in MCP-1 expression induced by MIF was inhibited by CD74- or CD44-specific siRNAs and SB203580, a p38 MAPK inhibitor. Further, CD74 or CD44 deficiency effectively inhibited MIF-induced p38 activation. MIF inhibitor ISO-1 reduced MCP-1 expression and p38 phosphorylation in CCl4-treated mouse liver. Our results showed that MIF regulates MCP-1 expression in hepatocytes of injured liver via CD74, CD44, and p38 MAPK in an autocrine manner, providing compelling information on the role of MIF in liver injury, and implying a new regulatory mechanism for liver inflammation. PMID:27273604

  13. Cholangiocarcinomas can originate from hepatocytes in mice

    PubMed Central

    Fan, Biao; Malato, Yann; Calvisi, Diego F.; Naqvi, Syed; Razumilava, Nataliya; Ribback, Silvia; Gores, Gregory J.; Dombrowski, Frank; Evert, Matthias; Chen, Xin; Willenbring, Holger

    2012-01-01

    Intrahepatic cholangiocarcinomas (ICCs) are primary liver tumors with a poor prognosis. The development of effective therapies has been hampered by a limited understanding of the biology of ICCs. Although ICCs exhibit heterogeneity in location, histology, and marker expression, they are currently thought to derive invariably from the cells lining the bile ducts, biliary epithelial cells (BECs), or liver progenitor cells (LPCs). Despite lack of experimental evidence establishing BECs or LPCs as the origin of ICCs, other liver cell types have not been considered. Here we show that ICCs can originate from fully differentiated hepatocytes. Using a mouse model of hepatocyte fate tracing, we found that activated NOTCH and AKT signaling cooperate to convert normal hepatocytes into biliary cells that act as precursors of rapidly progressing, lethal ICCs. Our findings suggest a previously overlooked mechanism of human ICC formation that may be targetable for anti-ICC therapy. PMID:22797301

  14. Overexpression of transforming growth factor-alpha causes liver enlargement and increased hepatocyte proliferation in transgenic mice.

    PubMed Central

    Webber, E. M.; Wu, J. C.; Wang, L.; Merlino, G.; Fausto, N.

    1994-01-01

    constitutive overexpression of TGF-alpha causes increased hepatocyte proliferation and liver enlargement in young animals and is associated with a delay in the establishment of hepatic polyploidy. These findings as well as the response of transgenic mice to partial hepatectomy show that constitutive overexpression of TGF-alpha initially caused increased but regulated hepatocyte proliferation which in older animals was compensated in part by a faster cell turnover. At 8 to 10 months of age, proliferative activity may become constitutive in some TGF-alpha expressing hepatocytes.(ABSTRACT TRUNCATED AT 400 WORDS) Images Figure 4 PMID:8053497

  15. Purification of Head-to-Tail-Type Regioregular Poly(3-hexylthiophene), HT-P3HexTh, and Investigation of the Effects of Polymer Purity on the Performance of Organic Field-Effect Transistors

    NASA Astrophysics Data System (ADS)

    Kokubo, Hisashi; Yamamoto, Takakazu; Kondo, Hiroshi; Akiyama, Yoshikazu; Fujimura, Itaru

    2003-10-01

    Analytically pure head-to-tail-type poly(3-hexylthiophene-2,5-diyl), HT-P3HexTh, has been obtained by the slow reprecipitation of the polymer. A FET made of purified HT-P3HexTh yielded a typical FET I-V curve with saturation and showed a five fold higher FET mobility than that of conventionally used HT-P3HexTh.

  16. Mechanisms of lysophosphatidylcholine-induced hepatocyte lipoapoptosis

    PubMed Central

    Kakisaka, Keisuke; Cazanave, Sophie C.; Fingas, Christian D.; Guicciardi, Maria E.; Bronk, Steven F.; Werneburg, Nathan W.; Mott, Justin L.

    2012-01-01

    Isolated hepatocytes undergo lipoapoptosis, a feature of hepatic lipotoxicity, on treatment with saturated free fatty acids (FFA) such as palmitate (PA). However, it is unknown if palmitate is directly toxic to hepatocytes or if its toxicity is indirect via the generation of lipid metabolites such as lysophosphatidylcholine (LPC). PA-mediated hepatocyte lipoapoptosis is associated with endoplasmic reticulum (ER) stress, c-Jun NH2-terminal kinase (JNK) activation, and a JNK-dependent upregulation of the potent proapoptotic BH3-only protein PUMA (p53 upregulated modulator of apoptosis). Our aim was to determine which of these mechanisms of lipotoxicity are activated by PA-derived LPC. We employed Huh-7 cells and isolated murine and human primary hepatocytes. Intracellular LPC concentrations increase linearly as a function of the exogenous, extracellular PA, stearate, or LPC concentration. Incubation of Huh-7 cells or primary hepatocytes with LPC induced cell death by apoptosis in a concentration-dependent manner. Substituting LPC for PA resulted in caspase-dependent cell death that was accompanied by activating phosphorylation of JNK with c-Jun phosphorylation and an increase in PUMA expression. LPC also induced ER stress as manifest by eIF2α phosphorylation and CAAT/enhancer binding homologous protein (CHOP) induction. LPC cytotoxicity was attenuated by pharmacological inhibition of JNK or glycogen synthase kinase-3 (GSK-3). Similarly, short-hairpin RNA (shRNA)-targeted knockdown of CHOP protected Huh-7 cells against LPC-induced toxicity. The LPC-induced PUMA upregulation was prevented by JNK inhibition or shRNA-targeted knockdown of CHOP. Finally, genetic deficiency of PUMA rendered murine hepatocytes resistant to LPC-induced apoptosis. We concluded that LPC-induced lipoapoptosis is dependent on mechanisms largely indistinguishable from PA. These data suggest that FFA-mediated cytotoxicity is indirect via the generation of the toxic metabolite, LPC. PMID:21995961

  17. H3K27me3 Does Not Orchestrate the Expression of Lineage-Specific Markers in hESC-Derived Hepatocytes In Vitro.

    PubMed

    Vanhove, Jolien; Pistoni, Mariaelena; Welters, Marc; Eggermont, Kristel; Vanslembrouck, Veerle; Helsen, Nicky; Boon, Ruben; Najimi, Mustapha; Sokal, Etienne; Collas, Philippe; Voncken, J Willem; Verfaillie, Catherine M

    2016-08-01

    Although pluripotent stem cells can be differentiated into the hepatocyte lineages, such cells retain an immature phenotype. As the chromatin state of regulatory regions controls spatiotemporal gene expression during development, we evaluated changes in epigenetic histone marks in lineage-specific genes throughout in vitro hepatocyte differentiation from human embryonic stem cells (hESCs). Active acetylation and methylation marks at promoters and enhancers correlated with progressive changes in gene expression. However, repression-associated H3K27me3 marks at these control regions showed an inverse correlation with gene repression during transition from hepatic endoderm to a hepatocyte-like state. Inhibitor of Enhancer of Zeste Homolog 2 (EZH2) reduced H3K27me3 decoration but did not improve hepatocyte maturation. Thus, H3K27me3 at regulatory regions does not regulate transcription and appears dispensable for hepatocyte lineage differentiation of hESCs in vitro. PMID:27477635

  18. Compatibility of Space Nuclear Power Plant Materials in an Inert He/Xe Working Gas Containing Reactive Impurities

    SciTech Connect

    MM Hall

    2006-01-31

    A major materials selection and qualification issue identified in the Space Materials Plan is the potential for creating materials compatibility problems by combining dissimilar reactor core, Brayton Unit and other power conversion plant materials in a recirculating, inert He/Xe gas loop containing reactive impurity gases. Reported here are results of equilibrium thermochemical analyses that address the compatibility of space nuclear power plant (SNPP) materials in high temperature impure He gas environments. These studies provide early information regarding the constraints that exist for SNPP materials selection and provide guidance for establishing test objectives and environments for SNPP materials qualification testing.

  19. Mn porphyrin-based SOD mimic, MnTnHex-2-PyP5+, and non-SOD mimic, MnTBAP3−, suppressed rat spinal cord ischemia/reperfusion injury via NF-κB pathways

    PubMed Central

    Celic, T.; Španjol, J.; Bobinac, M.; Tovmasyan, A.; Vukelic, I.; Reboucas, J. S.; Batinic-Haberle, I.; Bobinac, D.

    2015-01-01

    Herein we have demonstrated that both superoxide dismutase (SOD) mimic, cationic Mn(III) meso-tetrakis(N-n-hexylpyridinium-2-yl) porphyrin (MnTnHex-2-PyP5+), and non-SOD mimic, anionic Mn(III) meso-tetrakis(4-carboxylatophenyl)porphyrin (MnTBAP3−), protect against oxidative stress caused by spinal cord ischemia/reperfusion via suppression of nuclear factor kappa B (NF-κB) pro-inflammatory pathways. Earlier reports showed that Mn(III) N -alkylpyridylporphyrins were able to prevent the DNA binding of NF-κB in an aqueous system, whereas MnTBAP3− was not. Here, for the first time, in a complex in vivo system—animal model of spinal cord injury—a similar impact of MnTBAP3−, at a dose identical to that of MnTnHex-2-PyP5+, was demonstrated in NF-κB downregulation. Rats were treated subcutaneously at 1.5 mg/kg starting at 30 min before ischemia/reperfusion, and then every 12 h afterward for either 48 h or 7 days. The anti-inflammatory effects of both Mn porphyrins (MnPs) were demonstrated in the spinal cord tissue at both 48 h and 7 days. The down-regulation of NF-κ B, a major pro-inflammatory signaling protein regulating astrocyte activation, was detected and found to correlate well with the suppression of astrogliosis (as glial fibrillary acidic protein) by both MnPs. The markers of oxidative stress, lipid peroxidation and protein carbonyl formation, were significantly reduced by MnPs. The favorable impact of both MnPs on motor neurons (Tarlov score and inclined plane test) was assessed. No major changes in glutathione peroxidase- and SOD-like activities were demonstrated, which implies that none of the MnPs acted as SOD mimic. Increasing amount of data on the reactivity of MnTBAP3− with reactive nitrogen species (RNS) (·NO/HNO/ONOO−) suggests that RNS/MnTBAP3−-driven modification of NF-κB protein cysteines may be involved in its therapeutic effects. This differs from the therapeutic efficacy of MnTnHex-2-PyP5+ which presumably occurs via reactive

  20. Hepatocyte Growth Factor Reduces Free Cholesterol-Mediated Lipotoxicity in Primary Hepatocytes by Countering Oxidative Stress

    PubMed Central

    Domínguez-Pérez, Mayra; Nuño-Lámbarri, Natalia; Clavijo-Cornejo, Denise; Luna-López, Armando; Souza, Verónica; Bucio, Leticia; Miranda, Roxana U.; Muñoz, Linda; Gomez-Quiroz, Luis Enrique; Uribe-Carvajal, Salvador; Gutiérrez-Ruiz, María Concepción

    2016-01-01

    Cholesterol overload in the liver has shown toxic effects by inducing the aggravation of nonalcoholic fatty liver disease to steatohepatitis and sensitizing to damage. Although the mechanism of damage is complex, it has been demonstrated that oxidative stress plays a prominent role in the process. In addition, we have proved that hepatocyte growth factor induces an antioxidant response in hepatic cells; in the present work we aimed to figure out the protective effect of this growth factor in hepatocytes overloaded with free cholesterol. Hepatocytes from mice fed with a high-cholesterol diet were treated or not with HGF, reactive oxygen species present in cholesterol overloaded hepatocytes significantly decreased, and this effect was particularly associated with the increase in glutathione and related enzymes, such as γ-gamma glutamyl cysteine synthetase, GSH peroxidase, and GSH-S-transferase. Our data clearly indicate that HGF displays an antioxidant response by inducing the glutathione-related protection system. PMID:27143995

  1. The Zinc Transporter Zip14 Influences c-Met Phosphorylation and Hepatocyte Proliferation During Liver Regeneration in Mice

    PubMed Central

    AYDEMIR, TOLUNAY BEKER; SITREN, HARRY S.; COUSINS, ROBERT J.

    2013-01-01

    BACKGROUND & AIMS Zinc homeostasis in cells is maintained through tight regulation of zinc influx, efflux, and distribution to intracellular organelles by zinc transporters. The Zrt-Irt-like protein (ZIP) transporters facilitate zinc influx to the cytosol. Expression of the ZIP family member Zip14 can be induced by inflammatory cytokines, which also initiate liver regeneration. Hepatocyte proliferation is required for liver regeneration. Zinc regulates cell proliferation, tissue growth, and many mitogenic signaling pathways; we investigated its role in hepatocytes. METHODS Wild-type and Zip14−/− mice that underwent partial hepatectomy (70% of liver removed) were used as models of liver regeneration. We also analyzed AML12 hepatocytes that overexpressed Zip14. Proliferation was assessed with proliferating cell nuclear antigen, CD1, and Ki67 markers and along with assays of zinc content was related to protein tyrosine phosphatase 1B (PTP1B) and extracellular signal–regulated kinase 1/2 signaling. RESULTS Zip14 was up-regulated and hepatic zinc content increased during liver regeneration. Increased hepatic zinc inhibited activity of the phosphatase PTP1B and increased phosphorylation of c-Met, which promoted hepatocyte proliferation. AML12 cells that overexpressed Zip14 increased in zinc content and proliferation; PTP1B was inhibited and phosphorylation of c-Met increased. The increases in hepatic levels of zinc and hepatocyte proliferation that occurred following partial hepatectomy were not observed in Zip14−/− mice. CONCLUSIONS The transporter Zip14 mediates hepatic uptake of zinc during liver regeneration and for hepatocyte proliferation. These findings indicate that zinc transporter activity regulates liver tissue growth by sequestering zinc. Reagents that regulate ZIP14 activity might be developed as therapeutics to promote liver regeneration in patients with chronic liver disease. PMID:22374166

  2. Constrained spheroids for prolonged hepatocyte culture.

    PubMed

    Tong, Wen Hao; Fang, Yu; Yan, Jie; Hong, Xin; Hari Singh, Nisha; Wang, Shu Rui; Nugraha, Bramasta; Xia, Lei; Fong, Eliza Li Shan; Iliescu, Ciprian; Yu, Hanry

    2016-02-01

    Liver-specific functions in primary hepatocytes can be maintained over extended duration in vitro using spheroid culture. However, the undesired loss of cells over time is still a major unaddressed problem, which consequently generates large variations in downstream assays such as drug screening. In static culture, the turbulence generated by medium change can cause spheroids to detach from the culture substrate. Under perfusion, the momentum generated by Stokes force similarly results in spheroid detachment. To overcome this problem, we developed a Constrained Spheroids (CS) culture system that immobilizes spheroids between a glass coverslip and an ultra-thin porous Parylene C membrane, both surface-modified with poly(ethylene glycol) and galactose ligands for optimum spheroid formation and maintenance. In this configuration, cell loss was minimized even when perfusion was introduced. When compared to the standard collagen sandwich model, hepatocytes cultured as CS under perfusion exhibited significantly enhanced hepatocyte functions such as urea secretion, and CYP1A1 and CYP3A2 metabolic activity. We propose the use of the CS culture as an improved culture platform to current hepatocyte spheroid-based culture systems. PMID:26708088

  3. MicroRNA-21 accelerates hepatocyte proliferation in vitro via PI3K/Akt signaling by targeting PTEN

    SciTech Connect

    Yan-nan, Bai; Zhao-yan, Yu; Li-xi, Luo; Jiang, Yi; Qing-jie, Xia

    2014-01-17

    Highlights: •miRNAs-expression patterns of primary hepatocytes under proliferative status. •miR-21 expression level peaked at 12 h after stimulated by EGF. •miR-21 drive rapid S phase entry of primary hepatocytes. •PI3K/Akt signaling was modulated via targeting PTEN by miR-21. -- Abstract: MicroRNAs (miRNAs) are involved in controlling hepatocyte proliferation during liver regeneration. In this study, we established the miRNAs-expression patterns of primary hepatocytes in vitro under stimulation of epidermal growth factor (EGF), and found that microRNA-21 (miR-21) was appreciably up-regulated and peaked at 12 h. In addition, we further presented evidences indicating that miR-21 promotes primary hepatocyte proliferation through in vitro transfecting with miR-21 mimics or inhibitor. We further demonstrated that phosphatidylinositol 3′-OH kinase (PI3K)/Akt signaling was altered accordingly, it is, by targeting phosphatase and tensin homologue deleted on chromosome 10, PI3K/Akt signaling is activated by miR-21 to accelerate hepatocyte rapid S-phase entry and proliferation in vitro.

  4. Hydrodynamic Delivery of Cre Protein to Lineage-Mark or Time-Stamp Mouse Hepatocytes In situ

    PubMed Central

    Sonsteng, Katherine M.; Prigge, Justin R.; Talago, Emily A.; June, Ronald K.; Schmidt, Edward E.

    2014-01-01

    Cre-responsive fluorescent marker alleles are powerful tools for cell lineage tracing in mice; however their utility is limited by regulation of Cre activity. When targeting hepatocytes, hydrodynamic delivery of a Cre-expression plasmid can convert Cre-responsive alleles without inducing the intracellular or systemic antiviral responses often associated with viral-derived Cre-expression vectors. In this method, rapid high-volume intravenous inoculation induces hepatocyte-targeted uptake of extracellular molecules. Here we tested whether hydrodynamic delivery of Cre protein or Cre fused to the HIV-TAT cell-penetrating peptide could convert Cre-responsive reporters in hepatocytes of mice. Hydrodynamic delivery of 2 nmol of either Cre or TAT-Cre protein converted the reporter allele in 5 to 20% of hepatocytes. Neither protein gave detectable Cre activity in endothelia, non-liver organs, or non-hepatocyte cells in liver. Using mice homozygous for a Cre-responsive marker that directs red- (Cre-naïve) or green- (Cre-converted) fluorescent proteins to the nucleus, we assessed sub-saturation Cre-activity. One month after hydrodynamic inoculation with Cre protein, 58% of hepatocyte nuclei that were green were also red, indicating that less than half of the hepatocytes that had obtained enough Cre to convert one marker allele to green were able to convert all alleles. For comparison, one month after hydrodynamic delivery of a Cre-expression plasmid with a weak promoter, only 26% of the green nuclei were also red. Our results show that hydrodynamic delivery of Cre protein allows rapid allelic conversion in hepatocytes, but Cre-activity is sub-saturating so many cells will not convert multiple Cre-responsive alleles. PMID:24626158

  5. Lipid Rafts Establish Calcium Waves in Hepatocytes

    PubMed Central

    NAGATA, JUN; GUERRA, MATEUS T.; SHUGRUE, CHRISTINE A.; GOMES, DAWIDSON A.; NAGATA, NAOKI; NATHANSON, MICHAEL H.

    2010-01-01

    Background & Aims Polarity is critical for hepatocyte function. Ca2+ waves are polarized in hepatocytes because the inositol 1,4,5-trisphosphate receptor (InsP3R) is concentrated in the pericanalicular region, but the basis for this localization is unknown. We examined whether pericanalicular localization of the InsP3R and its action to trigger Ca2+ waves depends on lipid rafts. Methods Experiments were performed using isolated rat hepatocyte couplets and pancreatic acini, plus SkHep1 cells as nonpolarized controls. The cholesterol depleting agent methyl-beta-cyclodextrin (mβCD) was used to disrupt lipid rafts. InsP3R isoforms were examined by immunoblot and immunofluorescence. Ca2+ waves were examined by confocal microscopy. Results Type II InsP3Rs initially were localized to only some endoplasmic reticulum fractions in hepatocytes, but redistributed into all fractions in mβCD-treated cells. This InsP3R isoform was concentrated in the pericanalicular region, but redistributed throughout the cell after mβCD treatment. Vasopressin-induced Ca2+ signals began as apical-to-basal Ca2+ waves, and mβCD slowed the wave speed and prolonged the rise time. MβCD had a similar effect on Ca2+ waves in acinar cells but did not affect Ca2+ signals in SkHep1 cells, suggesting that cholesterol depletion has similar effects among polarized epithelia, but this is not a nonspecific effect of mβCD. Conclusions Lipid rafts are responsible for the pericanalicular accumulation of InsP3R in hepatocytes, and for the polarized Ca2+ waves that result. Signaling microdomains exist not only in the plasma membrane, but also in the nearby endoplasmic reticulum, which in turn, helps establish and maintain structural and functional polarity. PMID:17631147

  6. Back-side wear in HexLoc cups clinico-radiological, immunohistopathological, finite element, and retrieval analysis studies.

    PubMed

    Kawaji, Hiroyuki; Koistinen, Arto; Korhonen, Rami; Lappalainen, Reijo; Lohman, Martina; Soininen, Antti; Gomez Barrena, Enrique; Konttinen, Yrjo T; Ylinen, Pekka; Tallroth, Kaj

    2014-01-01

    The HexLoc locking system was designed to prevent back-side wear of the polyethylene liner in the modular cementless metal-backed acetabular cup, but failed. Back-side wear was analyzed using clinico-radiological data, immunohistopathology, finite element modeling (FEM, and retrieval analysis. Screw holes allowed entry of titanium oxide and exit of polyethylene particles. Birefringent polyethylene wear particles were found behind the metal cup in macrophages containing pro-inflammatory tumor necrosis factor-α and interleukin-1β, whereas fibroblast-like cells stained for osteoclastogenic receptor activator of nuclear factor kappa B ligand (RANKL). Computerized tomography revealed granulomas (83% versus 17 %) and cortical destruction (50% versus 5%) better than radiographs. In FEM, a change of the abduction angle from 45 to 60 deg, and liner thickness from 4.8 mm to 2.5 mm, increased the back-side wear by 90% and 120%, respectively. Screw holes were stress concentration areas; their removal decreased wear by 40%. Modeling results were validated in retrieved implants, which demonstrated extensive back-side wear damage of liners with a high abduction angle. Combined clinico-radiological, immunohistopathological, FEM, and retrieval analysis disclosed that back-side wear in the HexLoc design is sensitive to the abduction angle, liner thickness, and presence of screw holes. PMID:25747033

  7. Translational control plays a prominent role in the hepatocytic differentiation of HepaRG liver progenitor cells

    PubMed Central

    Parent, Romain; Beretta, Laura

    2008-01-01

    Background We investigated the molecular events associated with the differentiation of liver progenitor cells into functional and polarized hepatocytes, using human HepaRG cells that display potent hepatocytic differentiation-inducible properties and share some features with liver progenitor cells. Results Profiling of total and of polysome-bound transcripts isolated from HepaRG cells undergoing hepatocytic differentiation was performed. A group of 3,071 probe sets was reproducibly regulated by at least 2-fold in total or in polysome-bound RNA populations, upon differentiation. The fold changes in the total and the polysome-bound RNA populations for these 3,071 probe sets were poorly correlated (R = 0.38). Moreover, while the majority of the regulated polysome-bound RNA probe sets were up-regulated upon differentiation, the majority of the regulated probe sets selected from the total RNA population was down-regulated. Genes translationally up-regulated were associated with cell cycle inhibition, increased susceptibility to apoptosis and innate immunity. In contrast, genes transcriptionally up-regulated during differentiation corresponded in the majority to liver-enriched transcripts involved in lipid homeostasis and drug metabolism. Finally, several epithelial and hepato-specific transcripts were strongly induced in the total RNA population but were translationally repressed. Conclusion Translational regulation is the main genomic event associated with hepatocytic differentiation of liver progenitor cells in vitro and targets genes critical for moderating hepatocellular growth, cell death and susceptibility to pathogens. Transcriptional regulation targets specifically liver-enriched transcripts vital for establishing normal hepatic energy homeostasis, cell morphology and polarization. The hepatocytic differentiation is also accompanied by a reduction of the transcript content complexity. PMID:18221535

  8. Isolated rat hepatocytes can signal to other hepatocytes and bile duct cells by release of nucleotides.

    PubMed Central

    Schlosser, S F; Burgstahler, A D; Nathanson, M H

    1996-01-01

    Intercellular communication among certain cell types can occur via ATP secretion, which leads to stimulation of nucleotide receptors on target cells. In epithelial cells, however, intercellular communication is thought to occur instead via gap junctions. Here we examined whether one epithelial cell type, hepatocytes, can also communicate via nucleotide secretion. The effects on cytosolic Ca2+ ([Ca2+]i) of mechanical stimulation, including microinjection, were examined in isolated rat hepatocytes and in isolated bile duct units using confocal fluorescence video microscopy. Mechanical stimulation of a single hepatocyte evoked an increase in [Ca2+]i in the stimulated cell plus an unexpected [Ca2+]i rise in neighboring noncontacting hepatocytes. Perifusion with ATP before mechanical stimulation suppressed the [Ca2+]i increase, but pretreatment with phenylephrine did not. The P2 receptor antagonist suramin inhibited these intercellular [Ca2+]i signals. The ATP/ADPase apyrase reversibly inhibited the [Ca2+]i rise induced by mechanical stimulation, and did not block vasopressin-induced [Ca2+]i signals. Mechanical stimulation of hepatocytes also induced a [Ca2+]i increase in cocultured isolated bile duct units, and this [Ca2+]i increase was inhibited by apyrase as well. Finally, this form of [Ca2+]i signaling could be elicited in the presence of propidium iodide without nuclear labeling by that dye, indicating that this phenomenon does not depend on disruption of the stimulated cell. Thus, mechanical stimulation of isolated hepatocytes, including by microinjection, can evoke [Ca2+]i signals in the stimulated cell as well as in neighboring noncontacting hepatocytes and bile duct epithelia. This signaling is mediated by release of ATP or other nucleotides into the extracellular space. This is an important technical consideration given the widespread use of microinjection techniques for examining mechanisms of signal transduction. Moreover, the evidence provided suggests a

  9. p38α deficiency and oxidative stress cause cytokinesis failure in hepatocytes.

    PubMed

    Tormos Ana, M; Taléns-Visconti, Raquel; Jorques, María; Pérez-Garrido, Salvador; Bonora-Centelles, Ana; Nebreda Ángel, R; Sastre, Juan

    2014-10-01

    Cytokinesis is the last step in mitosis and it implies re-organization of the actin cytoskeleton. Its failure is one of the major mechanisms of polyploidy and binucleation in mammals. Our aims were 1) to assess the role of redox-sensitive p38α MAPK in cytokinesis by studying the liver of wild type mice or liver-specific p38α knock-out mice; 2) to assess the role of oxidative stress associated with hepatocyte isolation on cytokinesis. When p38α was down-regulated in hepatocytes, MK2 phosphorylation on threonine 334 was completely abrogated. Activation of MNK-1, required for abscission of the intercellular bridge, was diminished. Key proteins of the RhoA pathway (phospho-PRK2, nuclear phosphorylated cofilin, and cytosolic p27) were assessed confirming the impairment of this pathway. The absence of p38α in aging liver also led to a decrease in HSP27 phosphorylation, which is required for actin polymerization. Indeed, a severe impairment in the F-actin filamentous structure was found in the liver of old mice upon p38α deficiency. Consequently, long-term p38α MAPK down-regulation markedly affects the RhoA pathway and actin cytoskeleton dynamics inducing actin disassembly and cytokinesis failure upon aging. On the other hand, hepatocyte isolation caused marked glutathione depletion, increased generation of reactive oxygen species, and activated cell cycle entry. Addition of N-acetyl cysteine to isolation media prevented glutathione depletion, restrained the cell cycle entry, and abrogated defective cytokinesis and the formation of binucleated hepatocytes during isolation. Our results show that hepatocytes do enter into S phase but they do not complete cell division with age upon p38α deficiency or upon oxidative stress associated with isolation leading in both cases to cytokinesis failure and binucleation. PMID:26461300

  10. The emerging role of hepatocyte growth factor in renal diseases.

    PubMed

    Mao, Song; Zhang, Jianhua

    2016-06-01

    Hepatocyte growth factor (HGF), a kringle-containing polypeptide, acts on various epithelial cells to regulate cell growth, cell motility, and morphogenesis. HGF also accelerates tissue regeneration of injured organs and is regarded as a key molecule in organ regeneration. Besides the regeneration of the liver, HGF also plays a role in the renal regeneration. In addition, an adaptive alteration of HGF status in various renal diseases occurs. However, the precise role of HGF in various renal diseases remains elusive. The signaling pathways of HGF may be associated with renal diseases. In this review, we will try to provide an in-depth understanding of the underlying role of HGF and its possible interactions with other molecules in renal diseases. PMID:26460681

  11. ATF4 deficiency protects hepatocytes from oxidative stress via inhibiting CYP2E1 expression

    PubMed Central

    Wang, Chunxia; Li, Houkai; Meng, Qingshu; Du, Ying; Xiao, Fei; Zhang, Qian; Yu, Junjie; Li, Kai; Chen, Shanghai; Huang, Zhiying; Liu, Bin; Guo, Feifan

    2014-01-01

    Activating transcription factor (ATF) 4 is involved in the regulation of oxidative stress in fibroblasts and neurons. The role of ATF4 in hepatocytes, however, is unknown. The aim of this study was to investigate the role of ATF4 in hepatocytes in oxidative stress under a high-fat diet (HFD). Here, we showed that palmitate-stimulated reactive oxygen species (ROS) production and triglyceride (TG) accumulation is blocked by ATF4 deficiency in primary hepatocytes. Consistently, HFD-induced oxidative stress, TG accumulation and expression of cytochrome P450, family 2, subfamily, polypeptide 1 (CYP2E1) are also blocked by knocking down ATF4 expression in the mouse liver. This suggests that ATF4 might regulate oxidative stress viaCYP2E1 under an HFD. In addition, we observed that expression of CYP2E1 is indirectly regulated by ATF4 in a cAMP-responsive element binding protein (CREB)-dependent manner, which can directly activate the CYP2E1 promoter activity. Notably, ATF4-stimulated ROS production is inhibited in vivo by treatment with diallyl sulphide, a selective CYP2E1 inhibitor. Finally, we showed that ATF4 expression in the liver is responsible for the protective effects against HFD-induced CYP2E1 expression, oxidative stress, and TG accumulation. Taken together, these observations suggest that ATF4 is a novel regulator of oxidative stress as well as accumulation of TG in response to HFD. PMID:24373582

  12. Hyperinsulinemia is Associated with Increased Soluble Insulin Receptors Release from Hepatocytes.

    PubMed

    Hiriart, Marcia; Sanchez-Soto, Carmen; Diaz-Garcia, Carlos Manlio; Castanares, Diana T; Avitia, Morena; Velasco, Myrian; Mas-Oliva, Jaime; Macias-Silva, Marina; González-Villalpando, Clicerio; Delgado-Coello, Blanca; Sosa-Garrocho, Marcela; Vidaltamayo, Román; Fuentes-Silva, Deyanira

    2014-01-01

    It has been generally assumed that insulin circulates freely in blood. However it can also interact with plasma proteins. Insulin receptors are located in the membrane of target cells and consist of an alpha and beta subunits with a tyrosine kinase cytoplasmic domain. The ectodomain, called soluble insulin receptor (SIR) has been found elevated in patients with diabetes mellitus. We explored if insulin binds to SIRs in circulation under physiological conditions and hypothesize that this SIR may be released by hepatocytes in response to high insulin concentrations. The presence of SIR in rat and human plasmas and the culture medium of hepatocytes was explored using Western blot analysis. A purification protocol was performed to isolated SIR using affinity, gel filtration, and ion exchange chromatographies. A modified reverse hemolytic plaque assay was used to measure SIR release from cultured hepatocytes. Incubation with 1 nmol l(-1) insulin induces the release of the insulin receptor ectodomains from normal rat hepatocytes. This effect can be partially prevented by blocking protease activity. Furthermore, plasma levels of SIR were higher in a model of metabolic syndrome, where rats are hyperinsulinemic. We also found increased SIR levels in hyperinsulinemic humans. SIR may be an important regulator of the amount of free insulin in circulation. In hyperinsulinemia, the amount of this soluble receptor increases and this could lead to higher amounts of insulin bound to this receptor, rather than free insulin, which is the biologically active form of the hormone. This observation could enlighten the mechanisms of insulin resistance. PMID:24995000

  13. Changes in gluconeogenesis and intracellular lipid accumulation characterize uremic human hepatocytes ex vivo.

    PubMed

    Li, Meng; Ellis, Ewa; Johansson, Helene; Nowak, Greg; Isaksson, Bengt; Gnocchi, Davide; Parini, Paolo; Axelsson, Jonas

    2016-06-01

    It is well known that reduced glomerular filtration rate (GFR) leads to an increased risk of dyslipidemia, insulin resistance, and cardiovascular mortality. The liver is a central organ for metabolism, but its function in the uremic setting is still poorly characterized. We used human primary hepatocytes isolated from livers of nine donors with normal renal function to investigate perturbations in key metabolic pathways following exposure to uremic (n = 8) or healthy (n = 8) sera, and to serum-free control medium. Both uremic and healthy elicited consistent responses from hepatocytes from multiple donors and compared with serum-free control. However, at physiological insulin concentrations, uremic cells accumulated 56% more intracellular lipids. Also, when comparing uremic with healthy medium after culture, it contained more very-low-density lipoprotein-triglyceride and glucose. These changes were accompanied by decreased phosphorylation of AktS473 mRNA levels of key regulators of gluconeogenesis in uremic sera-treated hepatocytes such as phosphoenolpyruvate carboxykinase 1 and glucose 6-phosphate were elevated. We also found increased expression of 11β-hydroxysteroid dehydrogenase mRNA in uremic cells, along with high phosphorylation of downstream p53 and phospholipase C-γ1Y783 Thus our ex vivo data suggest that the uremic hepatocytes rapidly develop a glycogenic and lipogenic condition accompanied by perturbations in a large number of signaling networks. PMID:27056725

  14. Hepatocyte-specific Pten deficiency results in steatohepatitis and hepatocellular carcinomas

    PubMed Central

    Horie, Yasuo; Suzuki, Akira; Kataoka, Ei; Sasaki, Takehiko; Hamada, Koichi; Sasaki, Junko; Mizuno, Katsunori; Hasegawa, Go; Kishimoto, Hiroyuki; Iizuka, Masahiro; Naito, Makoto; Enomoto, Katsuhiko; Watanabe, Sumio; Mak, Tak Wah; Nakano, Toru

    2004-01-01

    PTEN is a tumor suppressor gene mutated in many human cancers, and its expression is reduced or absent in almost half of hepatoma patients. We used the Cre-loxP system to generate a hepatocyte-specific null mutation of Pten in mice (AlbCrePtenflox/flox mice). AlbCrePtenflox/flox mice showed massive hepatomegaly and steatohepatitis with triglyceride accumulation, a phenotype similar to human nonalcoholic steatohepatitis. Adipocyte-specific genes were induced in mutant hepatocytes, implying adipogenic-like transformation of these cells. Genes involved in lipogenesis and β-oxidation were also induced, possibly as a result of elevated levels of the transactivating factors PPARγ and SREBP1c. Importantly, the loss of Pten function in the liver led to tumorigenesis, with 47% of AlbCrePtenflox/flox livers developing liver cell adenomas by 44 weeks of age. By 74–78 weeks of age, 100% of AlbCrePtenflox/flox livers showed adenomas and 66% had hepatocellular carcinomas. AlbCrePtenflox/flox mice also showed insulin hypersensitivity. In vitro, AlbCrePtenflox/flox hepatocytes were hyperproliferative and showed increased hyperoxidation with abnormal activation of protein kinase B and MAPK. Pten is thus an important regulator of lipogenesis, glucose metabolism, hepatocyte homeostasis, and tumorigenesis in the liver. PMID:15199412

  15. Activation-dependent mitochondrial translocation of Foxp3 in human hepatocytes.

    PubMed

    Rojas, Joselyn; Teran-Angel, Guillermo; Barbosa, Luisa; Peterson, Darrell L; Berrueta, Lisbeth; Salmen, Siham

    2016-05-01

    Foxp3 is considered to be the master regulator for the development and function of regulatory T cells (Treg). Recently Foxp3, has been detected in extra lymphoid tissue, and in hepatocytes and has been associated with hepatocellular carcinoma (HCC), although its role has not been defined. Since it is expected that there is a relationship between protein localization, activity and cellular function, the aim of this study was to explore the subcellular localization of Foxp3 in resting and stimulated human hepatocytes. Foxp3 expression was measured by flow cytometry, subcellular fractioning, and immunofluorescence, and this data was used to track the shuttling of Foxp3 in different subcellular compartments in hepatocytes (HepG2 cell line), stimulated by using the PKC activators (PMA), core and preS1/2 antigen from hepatitis B virus (HBV). Our data shows that besides the nuclear location, mitochondrial translocation was detected after stimulation with PMA and at to a lesser extent, with preS1/2. In addition, Foxp3 is localizes at outer mitochondrial membrane. These results suggest a non-canonical role of Foxp3 in the mitochondrial compartment in human hepatocytes, and opens a new field about their role in liver damages during HBV infection. PMID:27068374

  16. Hyperinsulinemia is Associated with Increased Soluble Insulin Receptors Release from Hepatocytes

    PubMed Central

    Hiriart, Marcia; Sanchez-Soto, Carmen; Diaz-Garcia, Carlos Manlio; Castanares, Diana T.; Avitia, Morena; Velasco, Myrian; Mas-Oliva, Jaime; Macias-Silva, Marina; González-Villalpando, Clicerio; Delgado-Coello, Blanca; Sosa-Garrocho, Marcela; Vidaltamayo, Román; Fuentes-Silva, Deyanira

    2014-01-01

    It has been generally assumed that insulin circulates freely in blood. However it can also interact with plasma proteins. Insulin receptors are located in the membrane of target cells and consist of an alpha and beta subunits with a tyrosine kinase cytoplasmic domain. The ectodomain, called soluble insulin receptor (SIR) has been found elevated in patients with diabetes mellitus. We explored if insulin binds to SIRs in circulation under physiological conditions and hypothesize that this SIR may be released by hepatocytes in response to high insulin concentrations. The presence of SIR in rat and human plasmas and the culture medium of hepatocytes was explored using Western blot analysis. A purification protocol was performed to isolated SIR using affinity, gel filtration, and ion exchange chromatographies. A modified reverse hemolytic plaque assay was used to measure SIR release from cultured hepatocytes. Incubation with 1 nmol l−1 insulin induces the release of the insulin receptor ectodomains from normal rat hepatocytes. This effect can be partially prevented by blocking protease activity. Furthermore, plasma levels of SIR were higher in a model of metabolic syndrome, where rats are hyperinsulinemic. We also found increased SIR levels in hyperinsulinemic humans. SIR may be an important regulator of the amount of free insulin in circulation. In hyperinsulinemia, the amount of this soluble receptor increases and this could lead to higher amounts of insulin bound to this receptor, rather than free insulin, which is the biologically active form of the hormone. This observation could enlighten the mechanisms of insulin resistance. PMID:24995000

  17. A20 prevents chronic liver inflammation and cancer by protecting hepatocytes from death.

    PubMed

    Catrysse, L; Farhang Ghahremani, M; Vereecke, L; Youssef, S A; Mc Guire, C; Sze, M; Weber, A; Heikenwalder, M; de Bruin, A; Beyaert, R; van Loo, G

    2016-01-01

    An important regulator of inflammatory signalling is the ubiquitin-editing protein A20 that acts as a break on nuclear factor-κB (NF-κB) activation, but also exerts important cytoprotective functions. A20 knockout mice are cachectic and die prematurely due to excessive multi-organ inflammation. To establish the importance of A20 in liver homeostasis and pathology, we developed a novel mouse line lacking A20 specifically in liver parenchymal cells. These mice spontaneously develop chronic liver inflammation but no fibrosis or hepatocellular carcinomas, illustrating an important role for A20 in normal liver tissue homeostasis. Hepatocyte-specific A20 knockout mice show sustained NF-κB-dependent gene expression in the liver upon tumor necrosis factor (TNF) or lipopolysaccharide injection, as well as hepatocyte apoptosis and lethality upon challenge with sublethal doses of TNF, demonstrating an essential role for A20 in the protection of mice against acute liver failure. Finally, chronic liver inflammation and enhanced hepatocyte apoptosis in hepatocyte-specific A20 knockout mice was associated with increased susceptibility to chemically or high fat-diet-induced hepatocellular carcinoma development. Together, these studies establish A20 as a crucial hepatoprotective factor. PMID:27253414

  18. The effects of over expressing aquaporins on the cryopreservation of hepatocytes.

    PubMed

    Kumar, Balasubramanian K; Coger, Robin N; Schrum, Laura W; Lee, Charles Y

    2015-10-01

    During cryopreservation, aquaporins are critical in regulating water transport across cellular membranes and preventing osmotic damages. Hepatocytes express aquaporin (AQP) 0, 8, 9, 11, and 12; this study investigates whether increasing the localization of AQP8 on the cellular membrane would improve cell viability by increasing water transport during cryopreservation. Primary rat hepatocytes were cultured and treated with dibutyryl cAMP (Bt(2)cAMP) or glucagon to increase the expression of AQP8 at the cellular membrane via translocation. This phenomenon is verified through two experiments - confocal immunofluorescence microscopy and cell shrinkage analysis. The immunofluorescence results showed increase in AQP8 on the cellular membrane of treated cells, and cell shrinkage analysis showed an increase in water transport of treated cells compared to controls. Primary rat hepatocytes were treated with Bt(2)cAMP or glucagon and cryopreserved using standard protocols in a controlled rate freezer. This resulted in a significant increase in the cell viability on warming. These results indicate that Bt(2)cAMP or glucagon treated hepatocytes had increased expression of aquaporin in the cellular membrane, increased water transport during cryopreservation, and increased post-thaw viability. PMID:26247402

  19. Modulation of Insulin Sensitivity of Hepatocytes by the Pharmacological Downregulation of Phospholipase D

    PubMed Central

    Babenko, Nataliya A.; Kharchenko, Vitalina S.

    2015-01-01

    Background. The role of phospholipase D (PLD) as a positive modulator of glucose uptake activation by insulin in muscle and adipose cells has been demonstrated. The role of PLD in the regulation of glucose metabolism by insulin in the primary hepatocytes has been determined in this study. Methods. For this purpose, we studied effects of inhibitors of PLD on glucose uptake and glycogen synthesis stimulation by insulin. To determine the PLD activity, the method based on determination of products of transphosphatidylation reaction, phosphatidylethanol or phosphatidylbutanol, was used. Results. Inhibition of PLD by a general antagonist (1-butanol) or specific inhibitor, halopemide, or N-hexanoylsphingosine, or by cellular ceramides accumulated in doxorubicin-treated hepatocytes decreased insulin-stimulated glucose metabolism. Doxorubicin-induced hepatocytes resistance to insulin action could be abolished by inhibition of ceramide production. Halopemide could nullify this effect. Addition of propranolol, as well as inhibitors of phosphatidylinositol 3-kinase (PI3-kinase) (wortmannin, LY294002) or suppressors of Akt phosphorylation/activity, luteolin-7-O-glucoside or apigenin-7-O-glucoside, to the culture media could block cell response to insulin action. Conclusion. PLD plays an important role in the insulin signaling in the hepatocytes. PLD is activated downstream of PI3-kinase and Akt and is highly sensitive to ceramide content in the liver cells. PMID:26089893

  20. Generation of functional hepatocytes from human spermatogonial stem cells.

    PubMed

    Chen, Zheng; Sun, Min; Yuan, Qingqing; Niu, Minghui; Yao, Chencheng; Hou, Jingmei; Wang, Hong; Wen, Liping; Liu, Yun; Li, Zheng; He, Zuping

    2016-02-23

    To generate functional human hepatocytes from stem cells and/or extra-hepatic tissues could provide an important source of cells for treating liver diseases. Spermatogonial stem cells (SSCs) have an unlimited plasticity since they can dedifferentiate and transdifferentiate to other cell lineages. However, generation of mature and functional hepatocytes from human SSCs has not yet been achieved. Here we have for the first time reported direct transdifferentiation of human SSCs to mature and functional hepatocytes by three-step induction using the defined condition medium. Human SSCs were first transdifferentiated to hepatic stem cells, as evidenced by their morphology and biopotential nature of co-expressing hepatocyte and cholangiocyte markers but not hallmarks for embryonic stem cells. Hepatic stem cells were further induced to differentiate into mature hepatocytes identified by their morphological traits and strong expression of CK8, CK18, ALB, AAT, TF, TAT, and cytochrome enzymes rather than CK7 or CK19. Significantly, mature hepatocytes derived from human SSCs assumed functional attributes of human hepatocytes, because they could produce albumin, remove ammonia, and uptake and release indocyanine green. Moreover, expression of β-CATENIN, HNF4A, FOXA1 and GATA4 was upregulated during the transdifferentiation of human SSCs to mature hepatocytes. Collectively, human SSCs could directly transdifferentiate to mature and functional hepatocytes. This study could offer an invaluable source of human hepatocytes for curing liver disorders and drug toxicology screening and provide novel insights into mechanisms underlying human liver regeneration. PMID:26840458

  1. EXPERIMENTAL HEPATOCYTE XENOTRANSPLANTATION – A COMPREHENSIVE REVIEW OF THE LITERATURE

    PubMed Central

    Zhou, Huidong; Liu, Hong; Ezzelarab, Mohamed; Schmelzer, Eva; Wang, Yi; Gerlach, Jörg; Gridelli, Bruno; Cooper, David K. C.

    2015-01-01

    Background Hepatocyte transplantation is a potential therapy for certain diseases of the liver, including hepatic failure. However, there is a limited supply of human livers as a source of cells and, after isolation, human hepatocytes can be difficult to expand in culture, limiting the number available for transplantation. Hepatocytes from other species, e.g., the pig, have therefore emerged as a potential alternative source. We searched the literature through the end of 2014 to assess the current status of experimental research into hepatocyte xenotransplantation. Literature search and results The literature search identified 51 reports of in vivo cross-species transplantation of hepatocytes in a variety of experimental models. Most studies investigated the transplantation of human (n=23) or pig (n=19) hepatocytes. No studies explored hepatocytes from genetically-engineered pigs. The spleen was the most common site of transplantation (n=23), followed by the liver (through the portal vein [n=6]) and peritoneal cavity (n=19). In 47 studies (92%), there was evidence of hepatocyte engraftment and function across a species barrier. Conclusions The data provided by this literature search strengthen the hypothesis that xenotransplantation of hepatocytes is feasible and potentially successful as a clinical therapy for certain liver diseases, including hepatic failure. By excluding vascular structures, hepatocytes isolated from genetically-engineered pig livers may address some of the immunological problems of xenotransplantation. PMID:25950141

  2. Generation of functional hepatocytes from human spermatogonial stem cells

    PubMed Central

    Chen, Zheng; Sun, Min; Yuan, Qingqing; Niu, Minghui; Yao, Chencheng; Hou, Jingmei; Wang, Hong; Wen, Liping; Liu, Yun; Li, Zheng; He, Zuping

    2016-01-01

    To generate functional human hepatocytes from stem cells and/or extra-hepatic tissues could provide an important source of cells for treating liver diseases. Spermatogonial stem cells (SSCs) have an unlimited plasticity since they can dedifferentiate and transdifferentiate to other cell lineages. However, generation of mature and functional hepatocytes from human SSCs has not yet been achieved. Here we have for the first time reported direct transdifferentiation of human SSCs to mature and functional hepatocytes by three-step induction using the defined condition medium. Human SSCs were first transdifferentiated to hepatic stem cells, as evidenced by their morphology and biopotential nature of co-expressing hepatocyte and cholangiocyte markers but not hallmarks for embryonic stem cells. Hepatic stem cells were further induced to differentiate into mature hepatocytes identified by their morphological traits and strong expression of CK8, CK18, ALB, AAT, TF, TAT, and cytochrome enzymes rather than CK7 or CK19. Significantly, mature hepatocytes derived from human SSCs assumed functional attributes of human hepatocytes, because they could produce albumin, remove ammonia, and uptake and release indocyanine green. Moreover, expression of β-CATENIN, HNF4A, FOXA1 and GATA4 was upregulated during the transdifferentiation of human SSCs to mature hepatocytes. Collectively, human SSCs could directly transdifferentiate to mature and functional hepatocytes. This study could offer an invaluable source of human hepatocytes for curing liver disorders and drug toxicology screening and provide novel insights into mechanisms underlying human liver regeneration. PMID:26840458

  3. Assessing the therapeutic potential of lab-made hepatocytes.

    PubMed

    Rezvani, Milad; Grimm, Andrew A; Willenbring, Holger

    2016-07-01

    Hepatocyte transplantation has potential as a bridge or even alternative to whole-organ liver transplantation. Because donor livers are scarce, realizing this potential requires the development of alternative cell sources. To be therapeutically effective, surrogate hepatocytes must replicate the complex function and ability to proliferate of primary human hepatocytes. Ideally, they are also autologous to eliminate the need for immune suppression, which can have severe side effects and may not be sufficient to prevent rejection long term. In the past decade, several methods have been developed to generate hepatocytes from other readily and safely accessible somatic cells. These lab-made hepatocytes show promise in animal models of liver diseases, supporting the feasibility of autologous liver cell therapies. Here, we review recent preclinical studies exemplifying different types of lab-made hepatocytes that can potentially be used in autologous liver cell therapies. To define the therapeutic efficacy of current lab-made hepatocytes, we compare them to primary human hepatocytes, focusing on engraftment efficiency and posttransplant proliferation and function. In addition to summarizing published results, we discuss animal models and assays effective in assessing therapeutic efficacy. This analysis underscores the therapeutic potential of current lab-made hepatocytes, but also highlights deficiencies and uncertainties that need to be addressed in future studies aimed at developing liver cell therapies with lab-made hepatocytes. (Hepatology 2016;64:287-294). PMID:27014802

  4. Functional testing of hepatocytes following their recovery from cryopreservation.

    PubMed

    Innes, G K; Fuller, B J; Hobbs, K E

    1988-02-01

    Various tests of function have been suggested for assessing hepatocytes recovered from cryopreservation. In this study we have investigated hepatocyte attachment during tissue culture and cellular density in order to assess function and compared them with two classical dye exposure tests. The ability of hepatocytes to exclude trypan blue dye (TB) and metabolize fluorescein diacetate (FDA) was demonstrated. In populations of freshly prepared hepatocytes 88.07% were able to exclude TB and 87.31% were able to metabolize FDA. However in populations of hepatocytes recovered after cryopreservation using 1.5 M dimethyl sulfoxide as cryoprotectant only 33.44% were able to exclude TB and 31.59% able to metabolize FDA. Both of these tests gave the same estimate of functional ability. Density gradient centrifugation of hepatocytes on Percoll 400 (Pharmacia, Uppsala, Sweden) separated two populations of hepatocytes; one (density ca.1.07 g/ml Percoll) in which most of the cells were able to exclude TB and the second (density ca. 1.02 g/ml Percoll) in which they were stained blue. The dense population was highly enriched in dye-excluding hepatocytes: freshly prepared hepatocytes, 92.4%, and cryopreserved hepatocytes, 88.66%. When samples of these cells (2 x 10(6) dye-excluding cells per dish) were tested for their ability to attach to tissue culture dishes only 17.28% of the cryopreserved hepatocytes were able to attach compared to 55.28% of the freshly prepared cells. We conclude that cryopreservation of hepatocytes produces a population of cells which are not metabolically identical to a population of freshly prepared hepatocytes even though they appear to have the same buoyant density and dye-excluding capabilities.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3349809

  5. Phosphorylation dynamics of radixin in hypoxia-induced hepatocyte injury.

    PubMed

    Suda, Jo; Rockey, Don C; Karvar, Serhan

    2015-02-15

    The most prominent ezrin-radixin-moesin protein in hepatocytes is radixin, which is localized primarily at the canalicular microvilli and appears to be important in regulation of cell polarity and in localizing the multidrug resistance-associated protein 2 (Mrp-2) function. Our aim was to investigate how hypoxia affects radixin distribution and Mrp-2 function. We created wild-type and mutant constructs (in adenoviral vectors), which were expressed in WIF-B cells. The cellular distribution of Mrp-2 and radixin was visualized by fluorescence microscopy, and a 5-chloromethylfluorescein diacetate (CMFDA) assay was used to measure Mrp-2 function. Under usual conditions, cells infected with wild-type radixin, nonphosphorylatable radixin-T564A, and radixin-T564D (active phospho-mimicking mutant) were found to be heavily expressed in canalicular membrane compartment vacuoles, typically colocalizing with Mrp-2. In contrast, after hypoxia for 24 h, both endogenous and overexpressed wild-type radixin and the radixin-T564A mutant were found to be translocated to the cytoplasmic space. However, distribution of the radixin-T564D mutant, which mimics constant phosphorylation, was remarkably different, being associated with canalicular membranes even in hypoxic conditions. This dominant-active construct also prevented dissociation of radixin from the plasma membrane. Hypoxia also led to Mrp-2 mislocalization and caused Mrp-2 to be dissociated from radixin; the radixin phospho-mimicking mutant (T564D) abrogated this effect of hypoxia. Finally, hypoxia diminished the secretory response (measured using the CMFDA assay) in WIF-B cells, and the dominant-active construct (radixin-T567D) rescued this phenotype. Taken collectively, these findings suggest that radixin regulates Mrp-2 localization and function in hepatocytes and is important in hypoxic liver injury. PMID:25501552

  6. Metabolism of lipoproteins by human fetal hepatocytes

    SciTech Connect

    Carr, B.R.

    1987-12-01

    The rate of clearance of lipoproteins from plasma appears to play a role in the development of atherogenesis. The liver may account for as much as two thirds of the removal of low-density lipoprotein and one third of the clearance of high-density lipoprotein in certain animal species and humans, mainly by receptor-mediated pathways. The purpose of the present investigation was to determine if human fetal hepatocytes maintained in vitro take up and degrade lipoproteins. We first determined that the maximal binding capacity of iodine 125-iodo-LDL was approximately 300 ng of low-density lipoprotein protein/mg of membrane protein and an apparent dissociation constant of approximately 60 micrograms low-density lipoprotein protein/ml in membranes prepared from human fetal liver. We found that the maximal uptake of (/sup 125/I)iodo-LDL and (/sup 125/I)iodo-HDL by fetal hepatocytes occurred after 12 hours of incubation. Low-density lipoprotein uptake preceded the appearance of degradation products by 4 hours, and thereafter the degradation of low-density lipoprotein increased linearly for at least 24 hours. In contrast, high-density lipoprotein was not degraded to any extent by fetal hepatocytes. (/sup 125/I)Iodo-LDL uptake and degradation were inhibited more than 75% by preincubation with low-density lipoprotein but not significantly by high-density lipoprotein, whereas (/sup 125/I)iodo-HDL uptake was inhibited 70% by preincubation with high-density lipoprotein but not by low-density lipoprotein. In summary, human fetal hepatocytes take up and degrade low-density lipoprotein by a receptor-mediated process similar to that described for human extrahepatic tissues.

  7. Application of electroimmunoassay to the study of plasma protein synthesis in cultured hepatocytes.

    PubMed

    Grieninger, G; Pindyck, J; Hertzberg, K M; Mosesson, M W

    1979-01-01

    Electroimmunoassay has been applied to the study of plasma protein synthesis and secretion in liver cell cultures. The assay is performed on unconcentrated samples of culture medium containing the secreted plasma proteins and yields results within 2 hours. The characteristics of plasma protein production by the cultured hepatocytes coupled with the sensitivity of this assay permit the study of plasma protein in synthesis and its regulation by hormones and other agents without the routine use of radioisotopes. PMID:518014

  8. Crystal structure of 2,4,6-tris­(cyclo­hex­yloxy)-1,3,5-triazine

    PubMed Central

    Sankolli, Ravish; Hauser, Jürg; Row, T. N. Guru; Hulliger, Jürg

    2015-01-01

    The title compound, C21H33N3O3, is a tri-substituted cyclo­hex­yloxy triazine. In the crystal, the triazine rings form (C3i-PU) Piedfort units. The inter-centroid distance of the π–π inter­action involving the triazine rings is 3.3914 (10) Å. In the crystal, mol­ecules are linked by C—H⋯O hydrogen bonds, forming ribbons propagating along [1-10]. There are also weak C—H⋯N and C—H⋯O contacts present, linking inversion-related ribbons, forming a three-dimensional structure. PMID:26594503

  9. Incomplete inversion and double-valued fundamental linewidth of infrared HeNe and HeXe lasers

    SciTech Connect

    Kuppens, S.J.M.; Eijkelenborg, M.A. van; Schrama, C.A.; Exter, M.P. van; Woerdman, J.P.

    1996-03-01

    The authors have measured the quantum-limited linewidths of small HeNe 3.39 {micro}m and HeXe 3.51 {micro}m lasers. In contrast to the expected Schawlow-Townes behavior strong deviations from the inverse power dependence are observed, leading to a double-valued relation between the linewidth and the output power. This phenomenon is analyzed in terms of the increase of spontaneous emission, by a factor N{sub sp}, due to incompleteness of the inversion. The analysis shows that typically N{sub sp} has a value ranging from 1--10. Combining existing models for the pump power dependence of the level populations with measurements of the small signal gain they are able to explain the observed double-valued linewidth behavior in a quantitative way.

  10. A transient, Hex-Z nodal code corrected by discontinuity factors. Volume 1: The transient nodal code; Final report

    SciTech Connect

    Shatilla, Y.A.M.; Henry, A.F.

    1993-12-31

    This document constitutes Volume 1 of the Final Report of a three-year study supported by the special Research Grant Program for Nuclear Energy Research set up by the US Department of Energy. The original motivation for the work was to provide a fast and accurate computer program for the analysis of transients in heavy water or graphite-moderated reactors being considered as candidates for the New Production Reactor. Thus, part of the funding was by way of pass-through money from the Savannah River Laboratory. With this intent in mind, a three-dimensional (Hex-Z), general-energy-group transient, nodal code was created, programmed, and tested. In order to improve accuracy, correction terms, called {open_quotes}discontinuity factors,{close_quotes} were incorporated into the nodal equations. Ideal values of these factors force the nodal equations to provide node-integrated reaction rates and leakage rates across nodal surfaces that match exactly those edited from a more exact reference calculation. Since the exact reference solution is needed to compute the ideal discontinuity factors, the fact that they result in exact nodal equations would be of little practical interest were it not that approximate discontinuity factors, found at a greatly reduced cost, often yield very accurate results. For example, for light-water reactors, discontinuity factors found from two-dimensional, fine-mesh, multigroup transport solutions for two-dimensional cuts of a fuel assembly provide very accurate predictions of three-dimensional, full-core power distributions. The present document (volume 1) deals primarily with the specification, programming and testing of the three-dimensional, Hex-Z computer program. The program solves both the static (eigenvalue) and transient, general-energy-group, nodal equations corrected by user-supplied discontinuity factors.