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Sample records for hex regulates hepatocyte

  1. A hexA homologue from Photorhabdus regulates pathogenicity, symbiosis and phenotypic variation.

    PubMed

    Joyce, Susan A; Clarke, David J

    2003-03-01

    Photorhabdus is a genus of entomopathogenic Gram-negative bacteria that belong to the family Enterobactericeae. Remarkably, at the same time as being pathogenic to insect larvae, Photorhabdus also have a mutualistic relationship with entomophagous nematodes of the family Heterorhabditiae. Photorhabdus can be isolated in two phenotypically distinct forms, termed the primary and secondary variant. Both variants grow equally well and are equally virulent when injected into insect larvae. However, only the primary variant can colonize the intestinal tract of the IJ stage of the nematode and support nematode growth and development. The primary variant expresses several phenotypes that are absent from the secondary variant, including the production of extracellular enzymes, pigments, antibiotics and light. In this study, we use Photorhabdus temperata strain K122 to show that these primary-specific products are symbiosis factors, i.e. factors that are required for nematode growth and development. We also show that, in P. temperata K122, the production of these symbiosis factors is repressed in the secondary variant by the protein encoded by a gene with homology to hexA from Erwinia. Moreover, the derepression of the symbiosis factors in the secondary variant results in a significant attenuation of virulence to larvae of the greater wax moth, Galleria mellonella. This suggests that, during a normal infection, pathogenicity and symbiosis must be temporally separated and that HexA is involved in the regulation of this pathogen-symbiont transition.

  2. The hexA gene of Erwinia carotovora encodes a LysR homologue and regulates motility and the expression of multiple virulence determinants.

    PubMed

    Harris, S J; Shih, Y L; Bentley, S D; Salmond, G P

    1998-05-01

    We have identified a gene important for the regulation of exoenzyme virulence factor synthesis in the plant pathogen Erwinia carotovora ssp. carotovora (Ecc) and virulence and motility in Erwinia carotovora ssp. atroseptica (Eca). This gene, hexA (hyperproduction of exoenzymes), is a close relative of the Erwinia chrysanthemi (Echr) gene pecT and encodes a member of the LysR family of transcriptional regulators. hexA mutants in both Ecc and Eca produce abnormally high levels of the exoenzyme virulence factors pectate lyase, cellulase and protease. In addition, Eca hexA mutants show increased expression of the fliA and fliC genes and hypermotility. Consistent with a role as a global regulator, expression of hexA from even a low-copy plasmid can suppress exoenzyme production in Ecc and Eca and motility in Eca. Production of the quorum-sensing pheromone OHHL in Ecc hexA is higher throughout the growth curve compared with the wild-type strain. Overexpression of Ecc hexA also caused widespread effects in several strains of the opportunistic human pathogen, Serratia. Low-copy hexA expression resulted in repression of exoenzyme, pigment and antibiotic production and repression of the spreading phenotype. Finally, mutations in hexA were shown to increase Ecc or Eca virulence in planta.

  3. The Global Regulators Lrp, LeuO, and HexA Control Secondary Metabolism in Entomopathogenic Bacteria

    PubMed Central

    Engel, Yvonne; Windhorst, Carina; Lu, Xiaojun; Goodrich-Blair, Heidi; Bode, Helge B.

    2017-01-01

    Photorhabdus luminescens TTO1 and Xenorhabdus nematophila HGB081 are insect pathogenic bacteria and producers of various structurally diverse bioactive natural products. In these entomopathogenic bacteria we investigated the role of the global regulators Lrp, LeuO, and HexA in the production of natural products. Lrp is a general activator of natural product biosynthesis in X. nematophila and for most compounds in TTO1. Microarray analysis confirmed these results in X. nematophila and enabled the identification of additional biosynthesis gene clusters (BGC) regulated by Lrp. Moreover, when promoters of two X. nematophila BGC were analyzed, transcriptional activation by Lrp was observed. In contrast, LeuO in X. nematophila and P. luminescens has both repressing and activating features, depending on the natural product examined. Furthermore, heterologous overexpression of leuO from X. nematophila in the closely related Xenorhabdus szentirmaii resulted in overproduction of several natural products including novel compounds. The presented findings could be of importance for establishing a tool for overproduction of secondary metabolites and subsequent identification of novel compounds. PMID:28261170

  4. The Global Regulators Lrp, LeuO, and HexA Control Secondary Metabolism in Entomopathogenic Bacteria.

    PubMed

    Engel, Yvonne; Windhorst, Carina; Lu, Xiaojun; Goodrich-Blair, Heidi; Bode, Helge B

    2017-01-01

    Photorhabdus luminescens TTO1 and Xenorhabdus nematophila HGB081 are insect pathogenic bacteria and producers of various structurally diverse bioactive natural products. In these entomopathogenic bacteria we investigated the role of the global regulators Lrp, LeuO, and HexA in the production of natural products. Lrp is a general activator of natural product biosynthesis in X. nematophila and for most compounds in TTO1. Microarray analysis confirmed these results in X. nematophila and enabled the identification of additional biosynthesis gene clusters (BGC) regulated by Lrp. Moreover, when promoters of two X. nematophila BGC were analyzed, transcriptional activation by Lrp was observed. In contrast, LeuO in X. nematophila and P. luminescens has both repressing and activating features, depending on the natural product examined. Furthermore, heterologous overexpression of leuO from X. nematophila in the closely related Xenorhabdus szentirmaii resulted in overproduction of several natural products including novel compounds. The presented findings could be of importance for establishing a tool for overproduction of secondary metabolites and subsequent identification of novel compounds.

  5. 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

  6. 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…

  7. 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

  8. 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.

  9. Hepatocyte Growth Factor Regulates Angiotensin Converting Enzyme Expression*

    PubMed Central

    Day, Regina M.; Thiel, Gerald; Lum, Julie; Chévere, Rubén D.; Yang, Yongzhen; Stevens, Joanne; Sibert, Laura; Fanburg, Barry L.

    2008-01-01

    Hepatocyte growth factor (HGF) is a mitogen, morphogen, and motogen that functions in tissue healing and acts as an anti-fibrotic factor. The mechanism for this is not well understood. Recent studies implicate somatic angiotensin-converting enzyme (ACE) in fibrosis. We examined the effects of HGF on ACE expression in bovine pulmonary artery endothelial cells (BPAEC). Short term treatment of BPAEC with HGF transiently increased both ACE mRNA (3 h) and activity (24 h), as determined by ACE protease assays and reverse transcription-PCR. Incubation of BPAEC with HGF for longer periods suppressed ACE mRNA (6 h) and activity (72 h). In contrast, phorbol ester (PMA) treatment produced sustained increase in ACE mRNA and activity. We examined the short term molecular effects of HGF on ACE using PMA for comparison. HGF and PMA increased transcription from a luciferase reporter with the core ACE promoter, which contains a composite binding site for SP1/3 and Egr-1. Immunocytochemistry and electrophoretic mobility shift assay showed that both HGF and PMA increased Egr-1 binding. HGF also increased SP3 binding, as measured by EMSA. However, HGF and PMA increased the cellular activity of only Egr-1, not SP3, as measured by luciferase reporter assays. Deletion of the Egr-1 site in the reporter construct completely abrogated HGF-induced transcription but only ~50% of PMA-induced activity. Expression of dominant negative Egr-1 and SP3 blocked up-regulation of the ACE promoter by HGF but only reduced up-regulation by PMA. These results show that HGF transiently increases gene transcription of ACE via activation of Egr-1, whereas PMA regulation involves Egr-1 and additional factor(s). PMID:14679188

  10. hexA of Erwinia carotovora ssp. carotovora strain Ecc71 negatively regulates production of RpoS and rsmB RNA, a global regulator of extracellular proteins, plant virulence and the quorum-sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone.

    PubMed

    Mukherjee, A; Cui, Y; Ma, W; Liu, Y; Chatterjee, A K

    2000-04-01

    The soft-rotting bacterium, Erwinia carotovora ssp. carotovora (E. c. carotovora), produces an array of extracellular enzymes (= exoenzymes), including pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel) and protease (Prt), as well as HarpinEcc, the elicitor of hypersensitive reaction (HR). The production of these exoenzymes and HarpinEcc responds to plant products and the quorum-sensing signal [N-(3-oxohexanoyl)-L-homoserine lactone; OHL] and is subject to both transcriptional and post-transcriptional regulation. hexA of E. c. carotovora strain Ecc71 (hereafter hexA71), like that of another E. c. carotovora strain, negatively controls the production of exoenzymes, OHL and virulence in E. c. carotovora strain Ecc71. In addition to exoenzymes, HexA71 negatively regulates the expression of hrpNEcc, the structural gene for HarpinEcc. Exoenzyme overproduction is abolished by OHL deficiency in a HexA- and Ohll- double mutant, indicating that HexA and OHL are components of a common regulatory pathway controlling exoenzyme production. HexA71 negatively affects RpoS, as the levels of this alternative sigma factor are higher in the HexA- mutant than in the HexA+ strain. However, a HexA- and RpoS double mutant produces higher levels of exoenzymes and transcripts of pel-1, peh-1 and celVgenes than the HexA- and RpoS+ parent. Thus, the elevated levels of RpoS protein in the HexA- mutant do not account for exoenzyme overproduction. The following evidence associates for the first time the phenotypic changes in the HexA mutant to overproduction of rsmB RNA, a global regulator of exoenzymes, HarpinEcc, OHL and secondary metabolites. Analyses of rsmB transcripts and expression of an rsmB-lacZoperon fusion in E. c. carotovora strain Ecc71 revealed that HexA71 negatively regulates transcription of rsmB. Multiple copies of hexA71+ DNA suppress various phenotypes, including exoenzyme production in E. c. carotovora strain Ecc71, and concomitantly inhibit the production of rsm

  11. Hepatocytes in collagen sandwich: evidence for transcriptional and translational regulation

    PubMed Central

    1992-01-01

    The influence of extracellular matrix configuration on the tissue- specific function of cultured hepatocytes was investigated. Adult rat hepatocytes sandwiched between two layers of collagen gel were compared to cells cultured on a single layer of collagen gel for differences in the total RNA content, the level of albumin-specific mRNA, the rate of albumin gene transcription, and the rate of albumin mRNA translation. Adult hepatocytes in the sandwich system maintained the level of albumin mRNA similar to that found in the normal liver for at least six weeks, whereas the level of albumin mRNA declined rapidly in the single gel system. After one week of culture, hepatocytes in the single gel system could be induced to recover the high level of albumin mRNA and albumin production when a second layer of collagen gel was overlaid at that time. Furthermore, sandwiched hepatocytes maintained significantly higher transcriptional activity compared to cells in the single gel system. In addition to transcriptional control, the ultimate rate of albumin production was shown to depend on the rate of translation, which increased with culture time and reached a plateau in one to two weeks. This increase in translational activity over time in culture was observed in both the sandwich and the single gel systems and, thus, appeared to be independent of the configuration of extracellular matrix. PMID:1734019

  12. Role of Nuclear Constitutive Androstane Receptor in Regulation of Hepatocyte Proliferation and Hepatocarcinogenesis.

    PubMed

    Kazantseva, Y A; Pustylnyak, Y A; Pustylnyak, V O

    2016-04-01

    Activation of the constitutive androstane receptor (CAR) in hepatocytes occurs as a body adaptation in response to a number of external influences, and its functional activity is primarily related to induction of enzymes detoxifying xenobiotics. However, special attention was recently given to CAR due to the fact that its key role becomes unveiled in various physiological and pathophysiological processes occurring in the liver: gluconeogenesis, metabolism of fatty acids and bilirubin, hormonal regulation, proliferation of hepatocytes, and hepatocarcinogenesis. Here we review the main pathways and mechanisms that elevate hepatocyte proliferative activity related to CAR and whose disturbance may be a pivotal factor in hepatocarcinogenesis.

  13. p38α regulates actin cytoskeleton and cytokinesis in hepatocytes during development and aging

    PubMed Central

    Jorques, María; Rada, Patricia; Ramirez, Lorena; Valverde, Ángela M.; Nebreda, Ángel R.; Sastre, Juan

    2017-01-01

    Background Hepatocyte poliploidization is an age-dependent process, being cytokinesis failure the main mechanism of polyploid hepatocyte formation. Our aim was to study the role of p38α MAPK in the regulation of actin cytoskeleton and cytokinesis in hepatocytes during development and aging. Methods Wild type and p38α liver-specific knock out mice at different ages (after weaning, adults and old) were used. Results We show that p38α MAPK deficiency induces actin disassembly upon aging and also cytokinesis failure leading to enhanced binucleation. Although the steady state levels of cyclin D1 in wild type and p38α knock out old livers remained unaffected, cyclin B1- a marker for G2/M transition- was significantly overexpressed in p38α knock out mice. Our findings suggest that hepatocytes do enter into S phase but they do not complete cell division upon p38α deficiency leading to cytokinesis failure and binucleation. Moreover, old liver-specific p38α MAPK knock out mice exhibited reduced F-actin polymerization and a dramatic loss of actin cytoskeleton. This was associated with abnormal hyperactivation of RhoA and Cdc42 GTPases. Long-term p38α deficiency drives to inactivation of HSP27, which seems to account for the impairment in actin cytoskeleton as Hsp27-silencing decreased the number and length of actin filaments in isolated hepatocytes. Conclusions p38α MAPK is essential for actin dynamics with age in hepatocytes. PMID:28166285

  14. 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…

  15. The HexSim Model

    EPA Science Inventory

    HexSim version 2.0 is soon to be released by EPA's Western Ecology Division (WED). More than three years of work have gone into the development of this tool, which grew out of an EPA model called PATCH. HexSim makes it possible for non-programmers to develop sophisticated simula...

  16. 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.

  17. MiR-19a regulates PTEN expression to mediate glycogen synthesis in hepatocytes

    PubMed Central

    Dou, Lin; Meng, Xiangyu; Sui, Xiaofang; Wang, Shuyue; Shen, Tao; Huang, Xiuqing; Guo, Jun; Fang, Weiwei; Man, Yong; Xi, Jianzhong; Li, Jian

    2015-01-01

    MiR-19a, a member of mir-17-92 microRNA clusters, has been demonstrated to promote cell proliferation and angiogenesis via regulating the PI3K/AKT pathway, the major insulin signaling pathway. However, whether miR-19a plays an important role in glycogen synthesis in hepatocytes remains unknown. Here, we define the impact of miR-19a on glycogen synthesis and IL-6-induced reduced glycogenesis in hepatocytes and its underlying mechanisms. Our studies indicate that miR-19a was down-regulated in the livers of db/db mice and mice injected with IL-6, as well as mouse NCTC 1469 hepatocytes and HEP 1–6 hepatocytes treated by IL-6. We found that over-expression of miR-19a in NCTC 1469 cells and HEP 1–6 cells led to increased activation of the AKT/GSK pathway and synthesis of glycogen, whereas down-regulation of miR-19a impaired AKT/GSK phosphorylation and glycogenesis. Over-expression of miR-19a ameliorated IL-6-induced reduced glycogen synthesis in hepatocytes. Moreover, we identified PTEN as the target of miR-19a by a luciferase assay. Down-regulation of PTEN rescued the effects of miR-19a suppression on the activation of the AKT/GSK pathway and improved glycogenesis in NTC 1469 cells. These findings show for the first time that miR-19a might activate the AKT/GSK pathway and glycogenesis via down-regulation of PTEN expression. PMID:26111969

  18. 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.

  19. Regulatory network controlling extracellular proteins in Erwinia carotovora subsp. carotovora: FlhDC, the master regulator of flagellar genes, activates rsmB regulatory RNA production by affecting gacA and hexA (lrhA) expression.

    PubMed

    Cui, Yaya; Chatterjee, Asita; Yang, Hailian; Chatterjee, Arun K

    2008-07-01

    Erwinia carotovora subsp. carotovora produces an array of extracellular proteins (i.e., exoproteins), including plant cell wall-degrading enzymes and Harpin, an effector responsible for eliciting hypersensitive reaction. Exoprotein genes are coregulated by the quorum-sensing signal, N-acyl homoserine lactone, plant signals, an assortment of transcriptional factors/regulators (GacS/A, ExpR1, ExpR2, KdgR, RpoS, HexA, and RsmC) and posttranscriptional regulators (RsmA, rsmB RNA). rsmB RNA production is positively regulated by GacS/A, a two-component system, and negatively regulated by HexA (PecT in Erwinia chrysanthemi; LrhA [LysR homolog A] in Escherichia coli) and RsmC, a putative transcriptional adaptor. While free RsmA, an RNA-binding protein, promotes decay of mRNAs of exoprotein genes, binding of RsmA with rsmB RNA neutralizes the RsmA effect. In the course of studies of GacA regulation, we discovered that a locus bearing strong homology to the flhDC operon of E. coli also controls extracellular enzyme production. A transposon insertion FlhDC(-) mutant produces very low levels of pectate lyase, polygalacturonase, cellulase, protease, and E. carotovora subsp. carotovora Harpin (Harpin(Ecc)) and is severely attenuated in its plant virulence. The production of these exoproteins is restored in the mutant carrying an FlhDC(+) plasmid. Sequence analysis and transcript assays disclosed that the flhD operon of E. carotovora subsp. carotovora, like those of other enterobacteria, consists of flhD and flhC. Complementation analysis revealed that the regulatory effect requires functions of both flhD and flhC products. The data presented here show that FlhDC positively regulates gacA, rsmC, and fliA and negatively regulates hexA (lrhA). Evidence shows that FlhDC controls extracellular protein production through cumulative effects on hexA and gacA. Reduced levels of GacA and elevated levels of HexA in the FlhDC(-) mutant are responsible for the inhibition of rsmB RNA

  20. Regulation of activity and apical targeting of the Cl-/HCO3- exchanger in rat hepatocytes.

    PubMed Central

    Benedetti, A; Strazzabosco, M; Ng, O C; Boyer, J L

    1994-01-01

    To test the hypothesis that rat hepatocyte canalicular Cl-/HCO3- exchange activity might be regulated by HCO3- or protein kinase-induced changes in the apical targeting of vesicles, isolated rat hepatocytes were cultured in the presence or absence of HCO3-/CO2.Cl-/HCO3- exchange activity increased in cells cultured in the presence of HCO3-/CO2 or when stimulated by dibutyryl cAMP. Both of these effects were blocked by either colchicine or the protein kinase C agonist phorbol 12,13-dibutyrate. Fluorescence and confocal microscopy, respectively, revealed increased pericanalicular-apical membrane localization of two canalicular markers, peanut agglutinin and a 110-kDa canalicular ecto-ATPase, when hepatocyte couplets were preincubated in HCO3-/CO2-containing medium, an effect that was again blocked by colchicine. Dibutyryl cAMP also stimulated canalicular localization of the 110-kDa protein. These findings suggest that hepatocyte Cl-/HCO3- exchange activity is regulated by HCO3-/CO2 and by protein kinase A and protein kinase C agonists through microtubule-dependent targeting of vesicles containing this exchanger to the canalicular domain. Images Fig. 3 PMID:8290601

  1. Novel regulation of Ski protein stability and endosomal sorting by actin cytoskeleton dynamics in hepatocytes.

    PubMed

    Vázquez-Victorio, Genaro; Caligaris, Cassandre; Del Valle-Espinosa, Eugenio; Sosa-Garrocho, Marcela; González-Arenas, Nelly R; Reyes-Cruz, Guadalupe; Briones-Orta, Marco A; Macías-Silva, Marina

    2015-02-13

    TGF-β-induced antimitotic signals are highly regulated during cell proliferation under normal and pathological conditions, such as liver regeneration and cancer. Up-regulation of the transcriptional cofactors Ski and SnoN during liver regeneration may favor hepatocyte proliferation by inhibiting TGF-β signals. In this study, we found a novel mechanism that regulates Ski protein stability through TGF-β and G protein-coupled receptor (GPCR) signaling. Ski protein is distributed between the nucleus and cytoplasm of normal hepatocytes, and the molecular mechanisms controlling Ski protein stability involve the participation of actin cytoskeleton dynamics. Cytoplasmic Ski is partially associated with actin and localized in cholesterol-rich vesicles. Ski protein stability is decreased by TGF-β/Smads, GPCR/Rho signals, and actin polymerization, whereas GPCR/cAMP signals and actin depolymerization promote Ski protein stability. In conclusion, TGF-β and GPCR signals differentially regulate Ski protein stability and sorting in hepatocytes, and this cross-talk may occur during liver regeneration.

  2. Novel Regulation of Ski Protein Stability and Endosomal Sorting by Actin Cytoskeleton Dynamics in Hepatocytes*

    PubMed Central

    Vázquez-Victorio, Genaro; Caligaris, Cassandre; Del Valle-Espinosa, Eugenio; Sosa-Garrocho, Marcela; González-Arenas, Nelly R.; Reyes-Cruz, Guadalupe; Briones-Orta, Marco A.; Macías-Silva, Marina

    2015-01-01

    TGF-β-induced antimitotic signals are highly regulated during cell proliferation under normal and pathological conditions, such as liver regeneration and cancer. Up-regulation of the transcriptional cofactors Ski and SnoN during liver regeneration may favor hepatocyte proliferation by inhibiting TGF-β signals. In this study, we found a novel mechanism that regulates Ski protein stability through TGF-β and G protein-coupled receptor (GPCR) signaling. Ski protein is distributed between the nucleus and cytoplasm of normal hepatocytes, and the molecular mechanisms controlling Ski protein stability involve the participation of actin cytoskeleton dynamics. Cytoplasmic Ski is partially associated with actin and localized in cholesterol-rich vesicles. Ski protein stability is decreased by TGF-β/Smads, GPCR/Rho signals, and actin polymerization, whereas GPCR/cAMP signals and actin depolymerization promote Ski protein stability. In conclusion, TGF-β and GPCR signals differentially regulate Ski protein stability and sorting in hepatocytes, and this cross-talk may occur during liver regeneration. PMID:25561741

  3. 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

  4. Regulation of adiponectin receptor 1 in human hepatocytes by agonists of nuclear receptors

    SciTech Connect

    Neumeier, Markus; Weigert, Johanna; Schaeffler, Andreas; Weiss, Thomas; Kirchner, Stefan; Laberer, Sabine; Schoelmerich, Juergen; Buechler, Christa . E-mail: christa.buechler@klinik.uni-regensburg.de

    2005-09-02

    The adiponectin receptors AdipoR1 and AdipoR2 have been identified to mediate the insulin-sensitizing effects of adiponectin. Although AdipoR2 was suggested to be the main receptor for this adipokine in hepatocytes, AdipoR1 protein is highly abundant in primary human hepatocytes and hepatocytic cell lines. Nuclear receptors are main regulators of lipid metabolism and activation of peroxisome proliferator-activated receptor {alpha} and {gamma}, retinoid X receptor (RXR), and liver X receptor (LXR) by specific ligands may influence AdipoR1 abundance. AdipoR1 protein is neither altered by RXR or LXR agonists nor by pioglitazone. In contrast, fenofibric acid reduces AdipoR1 whereas hepatotoxic troglitazone upregulates AdipoR1 protein in HepG2 cells. Taken together this work shows for the first time that AdipoR1 protein is expressed in human hepatocytes but that it is not a direct target gene of nuclear receptors. Elevated AdipoR1 induced by hepatotoxic troglitazone may indicate a role of this receptor in adiponectin-mediated beneficial effects in liver damage.

  5. Involvement of hepatic stimulator substance in the regulation of hepatoblast maturation into hepatocytes in vitro.

    PubMed

    Sun, Guang-Yong; Dong, Ling-Yue; An, Wei

    2014-07-15

    Hepatic stimulator substance (HSS), also known as augmenter of liver regeneration (ALR), acts as a hepatotrophic growth factor to promote liver regeneration after liver damage or partial hepatectomy. However, the expression and function of HSS during liver development in mammals remain largely unknown. In this work, the hepatoblasts were isolated from mice at embryonic day 13.5 (E13.5), and HSS expression and its role during hepatoblast maturation were investigated. The results showed that HSS expression was enhanced in the hepatoblasts compared with mouse primary hepatocytes. HSS expression (23 kDa) was significantly decreased if the hepatoblast maturation was induced by a combination of oncostatin M (OSM), dexamethasone (DEX), and hepatocyte growth factor (HGF). We also found that knockdown of HSS expression (mainly 23-kDa isoform) by siRNA promoted hepatoblast maturation and also activated the signal transducer and activator of transcription 3 (STAT3) phosphorylation levels. However, if STAT3 activity was blocked by a small-molecule inhibitor Stattic, then hepatocyte maturation could be abolished, suggesting that STAT3 was most likely a potential molecule responsible for HSS signaling. In summary, our results demonstrated for the first time that HSS might be an active factor participating in the regulation of liver development and hepatocyte maturation.

  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 Central

    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.

    2012-01-01

    Summary 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. 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

  9. Differential regulation of cyclins D1 and D3 in hepatocyte proliferation.

    PubMed

    Rickheim, David G; Nelsen, Christopher J; Fassett, John T; Timchenko, Nikolai A; Hansen, Linda K; Albrecht, Jeffrey H

    2002-07-01

    Substantial evidence suggests that cyclin D1 plays a pivotal role in the control of the hepatocyte cell cycle in response to mitogenic stimuli, whereas the closely related protein cyclin D3 has not been extensively evaluated. In the current study, we examined the regulation of cyclins D1 and D3 during hepatocyte proliferation in vivo after 70% partial hepatectomy (PH) and in culture. In contrast to cyclin D1, which was nearly undetectable in quiescent liver and substantially up-regulated after PH, cyclin D3 was constitutively expressed and induced only modestly. In the regenerating liver, the concentration of cyclin D3 was only about 10% of that of cyclin D1. Cyclin D1 formed complexes primarily with cyclin-dependent kinase 4 (cdk4), which were markedly activated in the regenerating liver and readily sequestered the cell cycle inhibitory proteins, p21 and p27. Cyclin D3 bound to both cdk4 and cdk6. Cyclin D3/cdk6 activity was readily detectable in quiescent liver and changed little after PH, and this complex appeared to play a minor role in sequestering p21 and p27. In cultured hepatocytes, epidermal growth factor or insulin had little effect, but the combination of these agents substantially induced cyclin D1 and cell cycle progression. Inhibition of Mek1 or phosphoinositide 3-kinase markedly inhibited cyclin D1 expression and replication. In contrast, cyclin D3 was expressed in the absence of mitogens and was only modestly affected by these manipulations. In addition, growth-inhibitory extracellular matrix conditions inhibited cyclin D1 but not cyclin D3 expression. In conclusion, these results support the concept that cyclin D1 is critically regulated by extracellular stimuli that control proliferation, whereas cyclin D3 is regulated through different pathways and plays a distinct role in the liver.

  10. Regulation of alpha-1 acid glycoprotein synthesis by porcine hepatocytes in monolayer culture.

    PubMed

    Caperna, T J; Shannon, A E; Stoll, M; Blomberg, L A; Ramsay, T G

    2015-07-01

    Alpha-1 acid glycoprotein (AGP, orosomucoid, ORM-1) is a highly glycosylated mammalian acute-phase protein, which is synthesized primarily in the liver and represents the major serum protein in newborn pigs. Recent data have suggested that the pig is unique in that AGP is a negative acute-phase protein in this species, and its circulating concentration appears to be associated with growth rate. The purpose of the present study was to investigate the regulation of AGP synthesis in hepatocytes prepared from suckling piglets and to provide a framework to compare its regulation with that of haptoglobin (HP), a positive acute-phase protein. Hepatocytes were isolated from preweaned piglets and maintained in serum-free monolayer culture for up to 72 h. The influences of hormones, cytokines, and redox modifiers on the expression and secretion of AGP and HP were determined by relative polymerase chain reaction and by measuring the concentration of each protein secreted into culture medium. The messenger RNA abundance and/or secretion of AGP protein was enhanced by interleukin (IL)-17a, IL-1, and resveratrol and inhibited by tumor necrosis factor-α (TNF), oncostatin M, and thyroid hormone (P < 0.05). HP expression and synthesis were upregulated by oncostatin M, IL-6, and dexamethasone and downregulated by TNF (P < 0.01). The overall messenger RNA expression at 24 h was in agreement with the secreted protein patterns confirming that control of these proteins in hepatocytes is largely transcriptional. Moreover, these data support the consideration that AGP is a negative acute-phase reactant and appears to be regulated by cytokines (with the exception of TNF) and hormones primarily in a manner opposite to that of the positive acute-phase protein, HP.

  11. Differential regulation of constitutive androstane receptor expression by hepatocyte nuclear factor4alpha isoforms.

    PubMed

    Pascussi, Jean Marc; Robert, Agnes; Moreau, Amelie; Ramos, Jeanne; Bioulac-Sage, Paulette; Navarro, Francis; Blanc, Pierre; Assenat, Eric; Maurel, Patrick; Vilarem, Marie Jose

    2007-05-01

    Constitutive androstane receptor (CAR; NR1I3) controls the metabolism and elimination of endogenous and exogenous toxic compounds by up-regulating a battery of genes. In this work, we analyzed the expression of human CAR (hCAR) in normal liver during development and in hepatocellular carcinoma (HCC) and investigated the effect of hepatocyte nuclear factor 4alpha isoforms (HNF4alpha1 and HNF4alpha7) on the hCAR gene promoter. By performing functional analysis of hCAR 5'-deletions including mutants, chromatin immunoprecipitation in human hepatocytes, electromobility shift and cotransfection assays, we identified a functional and species-conserved HNF4alpha response element (DR1: ccAGGCCTtTGCCCTga) at nucleotide -144. Both HNF4alpha isoforms bind to this element with similar affinity. However, HNF4alpha1 strongly enhanced hCAR promoter activity whereas HNF4alpha7 was a poor activator and acted as a repressor of HNF4alpha1-mediated transactivation of the hCAR promoter. PGC1alpha stimulated both HNF4alpha1-mediated and HNF4alpha7-mediated hCAR transactivation to the same extent, whereas SRC1 exhibited a marked specificity for HNF4alpha1. Transduction of human hepatocytes by HNF4alpha7-expressing lentivirus confirmed this finding. In addition, we observed a positive correlation between CAR and HNF4alpha1 mRNA levels in human liver samples during development, and an inverse correlation between CAR and HNF4alpha7 mRNA levels in HCC. These observations suggest that HNF4alpha1 positively regulates hCAR expression in normal developing and adult livers, whereas HNF4alpha7 represses hCAR gene expression in HCC.

  12. Regulation of the Omega-3 Fatty Acid Biosynthetic Pathway in Atlantic Salmon Hepatocytes

    PubMed Central

    Ruyter, Bente; Berge, Gerd Marit; Sun, Yajing; Østbye, Tone-Kari Knutsdatter

    2016-01-01

    Limited availability of the n-3 fatty acids EPA and DHA have led to an interest in better understanding of the n-3 biosynthetic pathway and its regulation. The biosynthesis of alpha-linolenic acid to EPA and DHA involves several complex reaction steps including desaturation-, elongation- and peroxisomal beta-oxidation enzymes. The aims of the present experiments were to gain more knowledge on how this biosynthesis is regulated over time by different doses and fatty acid combinations. Hepatocytes isolated from salmon were incubated with various levels and combinations of oleic acid, EPA and DHA. Oleic acid led to a higher expression of the Δ6 fatty acid desaturase (fad) genes Δ6fad_a, Δ6fad_b, Δ6fad_c and the elongase genes elovl2 compared with cells cultured in medium enriched with DHA. Further, the study showed rhythmic variations in expression over time. Levels were reached where a further increase in specific fatty acids given to the cells not stimulated the conversion further. The gene expression of Δ6fad_a_and Δ6fad_b responded similar to fatty acid treatment, suggesting a co-regulation of these genes, whereas Δ5fad and Δ6fad_c showed a different regulation pattern. EPA and DHA induced different gene expression patterns, especially of Δ6fad_a. Addition of radiolabelled alpha-linolenic acid to the hepatocytes confirmed a higher degree of elongation and desaturation in cells treated with oleic acid compared to cells treated with DHA. This study suggests a complex regulation of the conversion process of n-3 fatty acids. Several factors, such as that the various gene copies are differently regulated, the gene expression show rhythmic variations and gene expression only affected to a certain level, determines when you get the maximum conversion of the beneficial n-3 fatty acids. PMID:27973547

  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. 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.

  15. 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

  16. 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...

  17. Autophagy regulates hepatocyte identity and epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions promoting Snail degradation.

    PubMed

    Grassi, G; Di Caprio, G; Santangelo, L; Fimia, G M; Cozzolino, A M; Komatsu, M; Ippolito, G; Tripodi, M; Alonzi, T

    2015-09-10

    Epithelial-to-mesenchymal transition (EMT) and the reverse process mesenchymal-to-epithelial transition (MET) are events involved in development, wound healing and stem cell behaviour and contribute pathologically to cancer progression. The identification of the molecular mechanisms underlying these phenotypic conversions in hepatocytes are fundamental to design specific therapeutic strategies aimed at optimising liver repair. The role of autophagy in EMT/MET processes of hepatocytes was investigated in liver-specific autophagy-deficient mice (Alb-Cre;ATG7(fl/fl)) and using the nontumorigenic immortalised hepatocytes cell line MMH. Autophagy deficiency in vivo reduces epithelial markers' expression and increases the levels of mesenchymal markers. These alterations are associated with an increased protein level of the EMT master regulator Snail, without transcriptional induction. Interestingly, we found that autophagy degrades Snail in a p62/SQSTM1 (Sequestosome-1)-dependent manner. Moreover, accordingly to a pro-epithelial function, we observed that autophagy stimulation strongly affects EMT progression, whereas it is necessary for MET. Finally, we found that the EMT induced by TGFβ affects the autophagy flux, indicating that these processes regulate each other. Overall, we found that autophagy regulates the phenotype plasticity of hepatocytes promoting their epithelial identity through the inhibition of the mesenchymal programme.

  18. Hepatocyte Factor JMJD5 Regulates Hepatitis B Virus Replication through Interaction with HBx

    PubMed Central

    Kouwaki, Takahisa; Okamoto, Toru; Ito, Ayano; Sugiyama, Yukari; Yamashita, Kazuo; Suzuki, Tatsuya; Kusakabe, Shinji; Hirano, Junki; Fukuhara, Takasuke; Yamashita, Atsuya; Saito, Kazunobu; Okuzaki, Daisuke; Watashi, Koichi; Sugiyama, Masaya; Yoshio, Sachiyo; Standley, Daron M.; Kanto, Tatsuya; Mizokami, Masashi; Moriishi, Kohji

    2016-01-01

    ABSTRACT Hepatitis B virus (HBV) is a causative agent for chronic liver diseases such as hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). HBx protein encoded by the HBV genome plays crucial roles not only in pathogenesis but also in replication of HBV. Although HBx has been shown to bind to a number of host proteins, the molecular mechanisms by which HBx regulates HBV replication are largely unknown. In this study, we identified jumonji C-domain-containing 5 (JMJD5) as a novel binding partner of HBx interacting in the cytoplasm. DNA microarray analysis revealed that JMJD5-knockout (JMJD5KO) Huh7 cells exhibited a significant reduction in the expression of transcriptional factors involved in hepatocyte differentiation, such as HNF4A, CEBPA, and FOXA3. We found that hydroxylase activity of JMJD5 participates in the regulation of these transcriptional factors. Moreover, JMJD5KO Huh7 cells exhibited a severe reduction in HBV replication, and complementation of HBx expression failed to rescue replication of a mutant HBV deficient in HBx, suggesting that JMJD5 participates in HBV replication through an interaction with HBx. We also found that replacing Gly135 with Glu in JMJD5 abrogates binding with HBx and replication of HBV. Moreover, the hydroxylase activity of JMJD5 was crucial for HBV replication. Collectively, these results suggest that direct interaction of JMJD5 with HBx facilitates HBV replication through the hydroxylase activity of JMJD5. IMPORTANCE HBx protein encoded by hepatitis B virus (HBV) plays important roles in pathogenesis and replication of HBV. We identified jumonji C-domain-containing 5 (JMJD5) as a novel binding partner to HBx. JMJD5 was shown to regulate several transcriptional factors to maintain hepatocyte function. Although HBx had been shown to support HBV replication, deficiency of JMJD5 abolished contribution of HBx in HBV replication, suggesting that HBx-mediated HBV replication is largely dependent on JMJD5. We showed that

  19. PI3K/AKT Signaling Regulates Bioenergetics in Immortalized Hepatocytes

    PubMed Central

    Li, Chen; Li, Yang; He, Lina; Agarwal, Amit R.; Zeng, Ni; Cadenas, Enrique; Stiles, Bangyan L.

    2013-01-01

    Regulation of cellular bioenergetics by PI3K/AKT signaling was examined in isogenic hepatocyte cell lines lacking the major inhibitor of PI3K/AKT signaling, PTEN (phosphatase and tensin homolog deleted on Chromosome 10). PI3K/AKT signaling was manipulated using the activator (IGF-1) and the inhibitor (LY 294002) of the PI3K/AKT pathway. Activation of PI3K/AKT signaling resulted in an enhanced anaerobic glycolysis and mitochondrial respiration. AKT, when phosphorylated and activated, translocated to mitochondria and localized within the membrane structure of mitochondria, where it phosphorylated a number of mitochondrial residence proteins including the subunits α and β of ATP synthase. Inhibition of GSK3β by either phosphorylation by AKT or lithium chloride resulted in activation of pyruvate dehydrogenase, i.e., decrease of its phosphorylated form. AKT-dependent phosphorylation of ATP synthase subunits α and β resulted in an increased complex activity. AKT translocation to mitochondria was associated with an increased expression and activity of complex I. These data suggest that the mitochondrial signaling pathway AKT-GSK3β-PDH, AKT-dependent phosphorylation of ATP synthase, and upregulation of mitochondrial complex I expression and activity are involved in the control of mitochondrial bioenergetics by increasing substrate availability and regulating the mitochondrial catalytic/energy-transducing capacity. PMID:23376468

  20. Hepatocyte nuclear factor 4α regulation of bile acid and drug metabolism

    PubMed Central

    Chiang, John YL

    2013-01-01

    The hepatocyte nuclear factor 4α (HNF4α) is a liver-enriched nuclear receptor that plays a critical role in early morphogenesis, fetal liver development, liver differentiation and metabolism. Human HNF4α gene mutations cause maturity on-set diabetes of the young type 1, an autosomal dominant non-insulin-dependent diabetes mellitus. HNF4α is an orphan nuclear receptor because of which the endogenous ligand has not been firmly identified. The trans-activating activity of HNF4α is enhanced by interacting with co-activators and inhibited by corepressors. Recent studies have revealed that HNF4α plays a central role in regulation of bile acid metabolism in the liver. Bile acids are required for biliary excretion of cholesterol and metabolites, and intestinal absorption of fat, nutrients, drug and xenobiotics for transport and distribution to liver and other tissues. Bile acids are signaling molecules that activate nuclear receptors to control lipids and drug metabolism in the liver and intestine. Therefore, HNF4α plays a central role in coordinated regulation of bile acid and xenobiotics metabolism. Drugs that specifically activate HNF4α could be developed for treating metabolic diseases such as diabetes, dyslipidemia and cholestasis, as well as drug metabolism and detoxification. PMID:19239393

  1. 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

  2. Heat-shock protein 70 modulates apoptosis signal-regulating kinase 1 in stressed hepatocytes of Mugil cephalus.

    PubMed

    Padmini, Ekambaram; Tharani, Jayachandran

    2014-10-01

    Oxidative stress causes damage at the cellular level and activates a number of signaling pathways. Heat-shock proteins (HSPs) play an important role in repair and protective mechanisms under cell response to stress conditions. HSP70 has been shown to act as an inhibitor of apoptosis. Apoptosis signal-regulating kinase-1 (ASK1) activity is regulated at multiple levels, one of which is through inhibition by cytosolic chaperons HSP70. The current study was aimed to investigate the alteration in signaling molecules that allow the fish to survive under stressed natural field conditions. The study also investigates the variation in biomolecular composition of hepatocytes by using Fourier transform infrared spectroscopy. The impact of stress on hepatocytes was assessed by measuring the level of lipid peroxides (LPO), catalase activity (CAT) and assessing the changes in hepatocytes of Mugil cephalus inhabiting Kovalam and Ennore estuaries. The expression of HSP70 and ASK1 were analyzed by immunoblot analysis and ELISA, respectively. The spectral analysis showed variations in biomolecular composition of hepatocytes at a wave number region of 4,000-400 cm(-1). There was significant decrease of CAT activity (p < 0.01) (25 %) with significant increase of LPO (p < 0.001) (35 %) and HSP70 (p < 0.001) and insignificant increase of ASK1 (p < 0.05) (16 %) in fish hepatocytes inhabiting Ennore estuary than Kovalam estuary. In conclusion, the present study suggests that the survival of fish in the Ennore estuary under stressed condition may be due to the upregulation of HSP70 that mediates the altered signal pathway which promotes cellular resistance against apoptosis.

  3. Microbiota regulate intestinal epithelial gene expression by suppressing the transcription factor Hepatocyte nuclear factor 4 alpha.

    PubMed

    Davison, James M; Lickwar, Colin R; Song, Lingyun; Breton, Ghislain; Crawford, Gregory E; Rawls, John F

    2017-04-06

    Microbiota influence diverse aspects of intestinal physiology and disease in part by controlling tissue-specific transcription of host genes. However, host genomic mechanisms mediating microbial control of intestinal gene expression are poorly understood. Hepatocyte nuclear factor 4 (HNF4) is the most ancient family of nuclear receptor transcription factors with important roles in human metabolic and inflammatory bowel diseases, but a role in host response to microbes is unknown. Using an unbiased screening strategy, we found that zebrafish Hnf4a specifically binds and activates a microbiota-suppressed intestinal epithelial transcriptional enhancer. Genetic analysis revealed that zebrafish hnf4a activates nearly half of the genes that are suppressed by microbiota, suggesting microbiota negatively regulate Hnf4a. In support, analysis of genomic architecture in mouse intestinal epithelial cells disclosed that microbiota colonization leads to activation or inactivation of hundreds of enhancers along with drastic genome-wide reduction of HNF4A and HNF4G occupancy. Interspecies meta-analysis suggested interactions between HNF4A and microbiota promote gene expression patterns associated with human inflammatory bowel diseases. These results indicate a critical and conserved role for HNF4A in maintaining intestinal homeostasis in response to microbiota.

  4. Advanced, enhanced HEX program for PIXE

    NASA Astrophysics Data System (ADS)

    Lipworth, A. D.; Annegarn, H. J.; Kneen, M. A.

    1993-04-01

    The REX code and subsequent HEX code, originating at Florida State University, have been extensively used for PIXE spectra fitting. In 1989 we produced a renovated HEX package: WITS-HEX, enabling the original Fortran program to be more accessible to the PIXE community. We modernised the user interface by replacing the batch mode of operation with an integrated, menu-driven environment. We added the ability to edit support data files from within the program, provided detailed feedback during the fitting process and enhanced spectral plots using high resolution colour graphics. Our prototype also permitted the inclusion of many more peaks and absorption coefficients into the element library than the original HEX, permitting a more extensive element request list to be used during the fitting operation. We have now completed the second phase of the renewal of HEX. The man-machine interface has been upgraded to conform to the IBM SAA Common User Access (CUA) standard. This eliminated several of the sequential (modal) human-computer dialogues, replacing them with a single parallel system. The support utility used in WITS-HEX to convert the binary format of spectra captured using foreign data acquisition systems has been replaced by code to directly access data in ASCII format. The program is now equipped with context-sensitive help and a tutorial. The polynomial background model has been supplemented by a digital filter model, eliminating the associated instability from the fitting process and other spectral features modelled. The program has been validated by comparing results with those obtained from the former versions: WITS-HEX and HEX. A demonstration version is available on request for evaluation purposes.

  5. Regulation of Hepatocyte Growth Factor in Mice with Pneumonia by Peptidases and Trans-Alveolar Flux

    PubMed Central

    Raymond, Wilfred W.; Xu, Xiang; Nimishakavi, Shilpa; Le, Catherine; McDonald, Donald M.; Caughey, George H.

    2015-01-01

    Hepatocyte growth factor (HGF) promotes lung epithelial repair after injury. Because prior studies established that human neutrophil proteases inactivate HGF in vitro, we predicted that HGF levels decrease in lungs infiltrated with neutrophils and that injury is less severe in lungs lacking HGF-inactivating proteases. After establishing that mouse neutrophil elastase cleaves mouse HGF in vitro, we tested our predictions in vivo by examining lung pathology and HGF in mice infected with Mycoplasma pulmonis, which causes neutrophilic tracheobronchitis and pneumonia. Unexpectedly, pneumonia severity was similar in wild type and dipeptidylpeptidase I-deficient (Dppi-/-) mice lacking neutrophil serine protease activity. To assess how this finding related to our prediction that Dppi-activated proteases regulate HGF levels, we measured HGF in serum, bronchoalveolar lavage fluid, and lung tissue from Dppi+/+ and Dppi-/- mice. Contrary to prediction, HGF levels were higher in lavage fluid from infected mice. However, serum and tissue concentrations were not different in infected and uninfected mice, and HGF lung transcript levels did not change. Increased HGF correlated with increased albumin in lavage fluid from infected mice, and immunostaining failed to detect increased lung tissue expression of HGF in infected mice. These findings are consistent with trans-alveolar flux rather than local production as the source of increased HGF in lavage fluid. However, levels of intact HGF from infected mice, normalized for albumin concentration, were two-fold higher in Dppi-/- versus Dppi+/+ lavage fluid, suggesting regulation by Dppi-activated proteases. Consistent with the presence of active HGF, increased expression of activated receptor c-Met was observed in infected tissues. These data suggest that HGF entering alveoli from the bloodstream during pneumonia compensates for destruction by Dppi-activated inflammatory proteases to allow HGF to contribute to epithelial repair. PMID

  6. Serine 249 phosphorylation by ATM protein kinase regulates hepatocyte nuclear factor-1α transactivation.

    PubMed

    Zhao, Long; Chen, Hui; Zhan, Yi-Qun; Li, Chang-Yan; Ge, Chang-Hui; Zhang, Jian-Hong; Wang, Xiao-Hui; Yu, Miao; Yang, Xiao-Ming

    2014-07-01

    Hepatocyte nuclear factor-1 alpha (HNF1α) exerts important effects on gene expression in multiple tissues. Several studies have directly or indirectly supported the role of phosphorylation processes in the activity of HNF1α. However, the molecular mechanism of this phosphorylation remains largely unknown. Using microcapillary liquid chromatography MS/MS and biochemical assays, we identified a novel phosphorylation site in HNF1α at Ser249. We also found that the ATM protein kinase phosphorylated HNF1α at Ser249 in vitro in an ATM-dependent manner and that ATM inhibitor KU55933 treatment inhibited phosphorylation of HNF1α at Ser249 in vivo. Coimmunoprecipitation assays confirmed the association between HNF1α and ATM. Moreover, ATM enhanced HNF1α transcriptional activity in a dose-dependent manner, whereas the ATM kinase-inactive mutant did not. The use of KU55933 confirmed our observation. Compared with wild-type HNF1α, a mutation in Ser249 resulted in a pronounced decrease in HNF1α transactivation, whereas no dominant-negative effect was observed. The HNF1αSer249 mutant also exhibited normal nuclear localization but decreased DNA-binding activity. Accordingly, the functional studies of HNF1αSer249 mutant revealed a defect in glucose metabolism. Our results suggested that ATM regulates the activity of HNF1α by phosphorylation of serine 249, particularly in glucose metabolism, which provides valuable insights into the undiscovered mechanisms of ATM in the regulation of glucose homeostasis.

  7. Insulin-Regulated Srebp-1c and Pck1 mRNA Expression in Primary Hepatocytes from Zucker Fatty but Not Lean Rats Is Affected by Feeding Conditions

    PubMed Central

    Zhang, Yan; Chen, Wei; Li, Rui; Li, Yang; Ge, Yuebin; Chen, Guoxun

    2011-01-01

    Insulin regulates the transcription of genes for hepatic glucose and lipid metabolism. We hypothesized that this action may be impaired in hepatocytes from insulin resistant animals. Primary hepatocytes from insulin sensitive Zucker lean (ZL) and insulin resistant Zucker fatty (ZF) rats in ad libitum or after an overnight fasting were isolated, cultured and treated with insulin and other compounds for analysis of gene expression using real-time PCR. The mRNA levels of one insulin-induced (Srebp-1c) and one insulin-suppressed (Pck1) genes in response to insulin, glucagon, and compactin treatments in hepatocytes from ad libitum ZL and ZF rats were analyzed. Additionally, the effects of insulin and T1317 on their levels in hepatocytes from ad libitum or fasted ZL or ZF rats were compared. The mRNA levels of Srebp-1c, Fas, and Scd1, but not that of Insr, Gck and Pck1, were higher in freshly isolated hepatocytes from ad libitum ZF than that from ZL rats. These patterns of Srebp-1c and Pck1 mRNA levels remained in primary hepatocyte cultured in vitro. Insulin's ability to regulate Srebp-1c and Pck1 expression was diminished in hepatocytes from ad libitum ZF, but not ZL rats. Glucagon or compactin suppressed Srebp-1c mRNA expression in lean, but not fatty hepatocytes. However, glucagon induced Pck1 mRNA expression similarly in hepatocytes from ad libitum ZL and ZF rats. Insulin caused the same dose-dependent increase of Akt phosphorylation in hepatocytes from ad libitum ZL and ZF rats. It synergized with T1317 to induce Srebp-1c, and suppressed Pck1 mRNA levels in hepatocytes from fasted, but not that from ad libitum ZF rats. We demonstrated that insulin was unable to regulate its downstream genes' mRNA expression in hepatocytes from ad libitum ZF rats. This impairment can be partially restored in hepatocytes from ZF rats after an overnight fasting, a phenomenon that deserves further investigation. PMID:21731709

  8. PNPLA3 is regulated by glucose in human hepatocytes, and its I148M mutant slows down triglyceride hydrolysis.

    PubMed

    Perttilä, Julia; Huaman-Samanez, Carolina; Caron, Sandrine; Tanhuanpää, Kimmo; Staels, Bart; Yki-Järvinen, Hannele; Olkkonen, Vesa M

    2012-05-15

    Liver fat is increased in carriers of the minor G allele in rs738409 (I148M amino acid substitution) in patatin-like phospholipase domain-containing 3 (PNPLA3)/adiponutrin. We studied transcriptional regulation of PNPLA3 in immortalized human hepatocytes (IHH) and human hepatoma cells (HuH7) and the impact of PNPLA3 I148M mutant on hepatocyte triglyceride metabolism. Studies in IHH showed that silencing of the carbohydrate response element-binding protein (ChREBP) abolished induction of PNPLA3 mRNA by glucose. Glucose-dependent binding of ChREBP to a newly identified carbohydrate response element in the PNPLA3 promoter was demonstrated by chromatin immunoprecipitation. Adenoviral overexpression of mouse ChREBP in IHH failed to induce PNPLA3 mRNA. [(3)H]acetate or [(3)H]oleate incorporation with 1-h pulse labeling or 18-h [(3)H]oleate labeling in HuH7 cells showed no effect of PNPLA3 I148M on triglyceride (TG) synthesis in the absence of free fatty acid (FFA) loading. Increased [(3)H]oleate accumulation into triglycerides in I148M-expressing cells was observed after 18 h of labeling in the presence of 200 μM FFA-albumin complexes. This was accompanied by increased PNPLA3 protein levels. The rate of hydrolysis of [(3)H]TG during lipid depletion was decreased significantly by PNPLA3 I148M. Our results suggest that PNPLA3 is regulated in human hepatocytes by glucose via ChREBP. PNPLA3 I148M enhances cellular accumulation of [(3)H]TG in the presence of excess FFA, which is known to stabilize PNPLA3 protein. These data do not exclude an effect of PNPLA3 I148M on hepatocyte lipogenesis but show that the mutant increases the stability of triglycerides.

  9. Metabolic regulation of fatty acid esterification and effects of conjugated linoleic acid on glucose homeostasis in pig hepatocytes.

    PubMed

    Conde-Aguilera, J A; Lachica, M; Nieto, R; Fernández-Fígares, I

    2012-02-01

    Conjugated linoleic acids (CLAs) are geometric and positional isomers of linoleic acid (LA) that promote growth, alter glucose metabolism and decrease body fat in growing animals, although the mechanisms are poorly understood. A study was conducted to elucidate the effects of CLA on glucose metabolism, triglyceride (TG) synthesis and IGF-1 synthesis in primary culture of porcine hepatocytes. In addition, hormonal regulation of TG and IGF-1 synthesis was addressed. Hepatocytes were isolated from piglets (n = 5, 16.0 ± 1.98 kg average body weight) by collagenase perfusion and seeded into collagen-coated T-25 flasks. Hepatocytes were cultured in William's E containing dexamethasone (10-8 and 10-7 M), insulin (10 and 100 ng/ml), glucagon (0 and 100 ng/ml) and CLA (1 : 1 mixture of cis-9, trans-11 and trans-10, cis-12 CLA, 0.05 and 0.10 mM) or LA (0.05 and 0.10 mM). Addition of CLA decreased gluconeogenesis (P < 0.05), whereas glycogen synthesis and degradation, TG synthesis and IGF-1 synthesis were not affected compared with LA. Increased concentration of fatty acids in the media decreased IGF-1 production (P < 0.001) and glycogen synthesis (P < 0.01), and increased gluconeogenesis (P < 0.001) and TG synthesis (P < 0.001). IGF-1 synthesis increased (P < 0.001) and TG synthesis decreased (P < 0.001) as dexamethasone concentration in the media rose. High insulin/glucagon increased TG synthesis. These results indicate that TG synthesis in porcine hepatocytes is hormonally regulated so that dexamethasone decreases and insulin/glucagon increases it. In addition, CLA decreases hepatic glucose production through decreased gluconeogenesis.

  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. S-Adenosylmethionine Regulates Dual-Specificity Mitogen-Activated Protein Kinase Phosphatase Expression in Mouse and Human Hepatocytes

    PubMed Central

    Tomasi, Maria Lauda; Ramani, Komal; Lopitz-Otsoa, Fernando; Rodríguez, Manuel S.; Li, Tony W. H.; Ko, Kwangsuk; Yang, Heping; Bardag-Gorce, Fawzia; Iglesias-Ara, Ainhoa; Feo, Francesco; Pascale, Maria Rosa; Mato, José M.; Lu, Shelly C.

    2010-01-01

    Increased mitogen-activated protein kinase (MAPK) activity correlates with a more malignant hepatocellular carcinoma (HCC) phenotype. There is a reciprocal regulation between p44/42 MAPK (extracellular signal-regulated kinase [ERK]1/2) and the dual-specificity MAPK phosphatase MKP-1/DUSP1. ERK phosphorylates DUSP1, facilitating its proteasomal degradation, whereas DUSP1 inhibits ERK activity. Methionine adenosyltransferase 1a (Mat1a) knockout (KO) mice express hepatic S-adenosylmethionine (SAM) deficiency and increased ERK activity and develop HCC. The aim of this study was to examine whether DUSP1 expression is regulated by SAM and if so, elucidate the molecular mechanisms. Studies were conducted using Mat1a KO mice livers, cultured mouse and human hepatocytes, and 20S and 26S proteasomes. DUSP1 messenger RNA (mRNA) and protein levels were reduced markedly in livers of Mat1a KO mice and in cultured mouse and human hepatocytes with protein falling to lower levels than mRNA. SAM treatment protected against the fall in DUSP1 mRNA and protein levels in mouse and human hepatocytes. SAM increased DUSP1 transcription, p53 binding to DUSP1 promoter, and stability of its mRNA and protein. Proteasomal chymotrypsin-like and caspase-like activities were increased in Mat1a KO livers and cultured hepatocytes, which was blocked by SAM treatment. SAM inhibited chymotrypsin-like and caspase-like activities by 40% and 70%, respectively, in 20S proteasomes and caused rapid degradation of some of the 26S proteasomal subunits, which was blocked by the proteasome inhibitor MG132. SAM treatment in Mat1a KO mice for 7 days raised SAM, DUSP1, mRNA and protein levels and lowered proteosomal and ERK activities. Conclusion DUSP1 mRNA and protein levels are lower in Mat1a KO livers and fall rapidly in cultured hepatocytes. SAM treatment increases DUSP1 expression through multiple mechanisms, and this may suppress ERK activity and malignant degeneration. PMID:20196119

  12. Biphasic Regulation of the NADPH Oxidase by HGF/c-Met Signaling Pathway in Primary Mouse Hepatocytes

    PubMed Central

    Clavijo-Cornejo, Denise; Enriquez-Cortina, Cristina; López-Reyes, Alberto; Domínguez-Pérez, Mayra; Nuño, Natalia; Domínguez-Meraz, Marcela; Bucio, Leticia; Souza, Verónica; Factor, Valentina M.; Thorgeirsson, Snorri S.; Gutiérrez-Ruiz, María Concepción; Gómez-Quiroz, Luis E.

    2013-01-01

    Redox signaling is emerging as an essential mechanism in the regulation of biological activities of the cell. The HGF/c-Met signaling pathway has been implicated as a key regulator of the cellular redox homeostasis and oxidative stress. We previously demonstrated that genetic deletion of c-met in hepatocytes disrupts redox homeostasis by a mechanism involving NADPH oxidase. Here, we were focused to address the mechanism of NADPH oxidase regulation by HGF/c-Met signaling in primary mouse hepatocytes and its relevance. HGF induced a biphasic mechanism of NADPH oxidase regulation. The first phase employed the rapid increase in production of ROS as signaling effectors to activate the Nrf2-mediated protective response resulting in up-regulation of the antioxidant proteins, such as NAD(P)H quinone oxidoreductase and γ-glutamylcysteine synthetase. The second phase operated under a prolonged HGF exposure, caused a suppression of the NADPH oxidase components, including NOX2, NOX4, p22 and p67, and was able to abrogate the TGFβ-induced ROS production and improve cell viability. In conclusion, HGF/c-Met induces a Nrf2-mediated protective response by a double mechanism driven by NADPH oxidase. PMID:23333744

  13. 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

  14. Regulation of hepatocyte proliferation. The feed-back system of hepatopoietin.

    PubMed

    Ruhenstroth-Bauer, G; Goldberg, M; Vogl, S

    1984-08-01

    After partial hepatectomy the low proliferation rate of hepatocytes increases dramatically. This is based on a feed-back system whose central link is a liver cell proliferation hormon, the so-called hepatopoietin. The hormon originates from the Peyer's patches: after their resection, the liver cell proliferation after partial hepatectomy decreases by about 80%. Hepatopoietin effects organ specific but species nonspecific.

  15. Regulation of hepatic microRNA expression by hepatocyte nuclear factor 4 alpha

    PubMed Central

    Lu, Hong; Lei, Xiaohong; Liu, Jerry; Klaassen, Curtis

    2017-01-01

    AIM To uncover the role of hepatocyte nuclear factor 4 alpha (HNF4α) in regulating hepatic expression of microRNAs. METHODS Microarray and real-time PCR were used to determine hepatic expression of microRNAs in young-adult mice lacking Hnf4α expression in liver (Hnf4α-LivKO). Integrative genomics viewer software was used to analyze the public chromatin immunoprecipitation-sequencing datasets for DNA-binding of HNF4α, RNA polymerase-II, and histone modifications to loci of microRNAs in mouse liver and human hepatoma cells. Dual-luciferase reporter assay was conducted to determine effects of HNF4α on the promoters of mouse and human microRNAs as well as effects of microRNAs on the untranslated regions (3’UTR) of two genes in human hepatoma cells. RESULTS Microarray data indicated that most microRNAs remained unaltered by Hnf4α deficiency in Hnf4α-LivKO mice. However, certain liver-predominant microRNAs were down-regulated similarly in young-adult male and female Hnf4α-LivKO mice. The down-regulation of miR-101, miR-192, miR-193a, miR-194, miR-215, miR-802, and miR-122 as well as induction of miR-34 and miR-29 in male Hnf4α-LivKO mice were confirmed by real-time PCR. Analysis of public chromatin immunoprecipitation-sequencing data indicates that HNF4α directly binds to the promoters of miR-101, miR-122, miR-194-2/miR-192 and miR-193, which is associated with histone marks of active transcription. Luciferase reporter assay showed that HNF4α markedly activated the promoters of mouse and human miR-101b/miR-101-2 and the miR-194/miR-192 cluster. Additionally, miR-192 and miR-194 significantly decreased activities of luciferase reporters for the 3’UTR of histone H3F3 and chromodomain helicase DNA binding protein 1 (CHD1), respectively, suggesting that miR-192 and miR-194 might be important in chromosome remodeling through directly targeting H3F3 and CHD1. CONCLUSION HNF4α is essential for hepatic basal expression of a group of liver-enriched micro

  16. Regulation of Bcl-2 and Bcl-xL anti-apoptotic protein expression by nuclear receptor PXR in primary cultures of human and rat hepatocytes.

    PubMed

    Zucchini, Nathalie; de Sousa, Georges; Bailly-Maitre, Béatrice; Gugenheim, Jean; Bars, Rémi; Lemaire, Géraldine; Rahmani, Roger

    2005-08-15

    The pregnane X receptor (PXR) plays a major role in the protection of the body by regulating the genes involved in the metabolism and elimination of potentially toxic xeno- and endobiotics. We previously described that PXR activator dexamethasone protects hepatocytes from spontaneous apoptosis. We hypothesise a PXR-dependent co-regulation process between detoxication and programmed cell death. Using primary cultured human and rat hepatocytes, we investigated to determine if PXR is implicated in the regulation of Bcl-2 and Bcl-xL, two crucial apoptosis inhibitors. In the present study we demonstrated that the treatment of primary cultured hepatocytes with PXR agonists increased hepatocyte viability and protects them from staurosporine-induced apoptosis. The anti-apoptotic capacity of PXR activation was correlated with Bcl-2 and Bcl-xL induction at both the transcriptional and protein levels in man and rats, respectively. The inhibition of PXR expression by antisense oligonucleotide abolished PXR activators Bcl-xL induction. Accordingly, PXR overexpression in HepG2 cells led to bcl-2 induction upon clotrimazole treatment and protects cells against Fas-induced apoptosis. Our results demonstrate that PXR expression is required for Bcl-2 and Bcl-xL up-regulation upon PXR activators treatment in human and rat hepatocytes. They also suggest that PXR may protect the liver against chemicals by simultaneously regulating detoxication and the apoptotic pathway.

  17. 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.

  18. Effect of aging on regulation of sdi-1 in rat hepatocytes.

    PubMed

    Kitano, S; Venable, S; Smith, J R; Reed, T D; Roth, G S

    1996-08-05

    We examined basal and EGF stimulated DNA synthesis as well as sdi-1 mRNA and protein in primary hepatocyte cultures, and basal levels of sdi-1 mRNA and protein in whole liver homogenates from 6 and 24 month old rats. Since EGF stimulated DNA synthesis decreases with age, it was hypothesized that basal and EGF stimulated levels of sdi-1 mRNA and protein, an inhibitor of DNA synthesis, might increase. Surprisingly, however both sdi-1 mRNA and protein actually decreased both in cells and homogenates of old rats. These results indicate that the age-related impairment in EGF stimulated DNA synthesis in hepatocytes appears to occur prior to or parallel with sdi-1 expression and cannot be explained on the basis of increased inhibition due to elevated levels of this protein.

  19. Hex death: voodoo magic or persuasion?

    PubMed

    Meador, C K

    1992-03-01

    Hex death is one that follows a ritualized pronouncement of death by a powerful authority, a phenomenon not widely accepted by the Western medical community, despite scattered accounts witnessed by reputable observers. Case histories of two patients are presented. The first patient, a poorly educated man near death after a hex pronounced by a local voodoo priest, rapidly recovered after ingenious words and actions by his family physician. The second, who had a diagnosis of metastatic carcinoma of the esophagus, died believing he was dying of widespread cancer, as did his family and his physicians. At autopsy, only a 2 cm nodule of cancer in his liver was found. The cases raise several intriguing questions. Is death from hexing limited to ignorant and superstitious tribes, or is it part of some general phenomenon basic to many forms of human communication? Is hex death only a form of human persuasion? If we can cause death by what we say or do, then what lesser behavior do we induce in our patients? Can diagnostic labels of serious or life-threatening diseases, whether correct or not, be part of this general phenomenon?

  20. 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.

  1. Protein inhibitor of activated STAT 4 (PIAS4) regulates pro-inflammatory transcription in hepatocytes by repressing SIRT1.

    PubMed

    Sun, Lina; Fan, Zhiwen; Chen, Junliang; Tian, Wenfang; Li, Min; Xu, Huihui; Wu, Xiaoyan; Fang, Mingming; Xia, Jun; Xu, Yong

    2016-07-12

    Excessive nutrition promotes the pathogenesis of non-alcoholic steatohepatitis (NASH), characterized by the accumulation of pro-inflammation mediators in the liver. In the present study we investigated the regulation of pro-inflammatory transcription in hepatocytes by protein inhibitor of activated STAT 4 (PIAS4) in this process and the underlying mechanisms. We report that expression of the class III deacetylase SIRT1 was down-regulated in the livers of NASH mice accompanied by a simultaneous increase in the expression and binding activity of PIAS4. Exposure to high glucose stimulated the expression PIAS4 in cultured hepatocytes paralleling SIRT1 repression. Estrogen, a known NASH-protective hormone, ameliorated SIRT1 trans-repression by targeting PIAS4. Over-expression of PIAS4 enhanced, while PIAS4 knockdown alleviated, repression of SIRT1 transcription by high glucose. Lentiviral delivery of short hairpin RNA (shRNA) targeting PIAS4 attenuated hepatic inflammation in NASH mice by restoring SIRT1 expression. Mechanistically, PIAS4 promoted NF-κB-mediated pro-inflammatory transcription in a SIRT1 dependent manner. In conclusion, our study indicates that PIAS4 mediated SIRT1 repression in response to nutrient surplus contributes to the pathogenesis of NASH. Therefore, targeting PIAS4 might provide novel therapeutic strategies in the intervention of NASH.

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

    PubMed

    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.

  3. Distribution dynamics and functional importance of NHERF1 in regulation of Mrp-2 trafficking in hepatocytes.

    PubMed

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

    2014-10-15

    Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) is a multifunctional scaffolding protein that interacts with receptors and ion transporters in its PDZ domains and with the ezrin-radixin-moesin (ERM) family of proteins in its COOH terminus. The role of NHERF1 in hepatocyte function remains largely unknown. We examine the distribution and physiological significance of NHERF1 and multidrug resistance-associated protein 2 (Mrp-2) in hepatocytes. A WT radixin binding site mutant (F355R) and NHERF1 PDZ1 and PDZ2 domain adenoviral mutant constructs were tagged with yellow fluorescent protein and expressed in polarized hepatocytes to study localization and function of NHERF1. Cellular distribution of NHERF1 and radixin was visualized by fluorescence microscopy. A 5-chloromethylfluorescein diacetate (CMFDA) assay was used to characterize Mrp-2 function. Similar to Mrp-2, WT NHERF1 and the NHERF1 PDZ2 deletion mutant were localized to the canalicular membrane. In contrast, the radixin binding site mutant (F355R) and the NHERF1 PDZ1 deletion mutant, which interacts poorly with Mrp-2, were rarely associated with the canalicular membrane. Knockdown of NHERF1 led to dramatically impaired CMFDA secretory response. Use of CMFDA showed that the NHERF1 PDZ1 and F355R mutants were devoid of a secretory response, while WT NHERF1-infected cells exhibited increased secretion of glutathione-methylfluorescein. The data indicate that NHERF1 interacts with Mrp-2 via the PDZ1 domain of NHERF1 and, furthermore, that NHERF1 is essential for maintaining the localization and function of Mrp-2.

  4. Commensal Bacteria-induced Interleukin 1β (IL-1β) Secreted by Macrophages Up-regulates Hepcidin Expression in Hepatocytes by Activating the Bone Morphogenetic Protein Signaling Pathway.

    PubMed

    Shanmugam, Nanda Kumar N; Chen, Kejie; Cherayil, Bobby J

    2015-12-18

    The liver hormone hepcidin is the central regulator of systemic iron metabolism. Its increased expression in inflammatory states leads to hypoferremia and anemia. Elucidation of the mechanisms that up-regulate hepcidin during inflammation is essential for developing rational therapies for this anemia. Using mouse models of inflammatory bowel disease, we have shown previously that colitis-associated hepcidin induction is influenced by intestinal microbiota composition. Here we investigate how two commensal bacteria, Bifidobacterium longum and Bacteroides fragilis, representative members of the gut microbiota, affect hepcidin expression. We found that supernatants of a human macrophage cell line infected with either of the bacteria up-regulated hepcidin when added to a human hepatocyte cell line. This activity was abrogated by neutralization of IL-1β. Moreover, purified IL-1β increased hepcidin expression when added to the hepatocyte line or primary human hepatocytes and when injected into mice. IL-1β activated the bone morphogenetic protein (BMP) signaling pathway in hepatocytes and in mouse liver, as indicated by increased phosphorylation of small mothers against decapentaplegic proteins. Activation of BMP signaling correlated with IL-1β-induced expression of BMP2 in human hepatocytes and activin B in mouse liver. Treatment of hepatocytes with two different chemical inhibitors of BMP signaling or with a neutralizing antibody to BMP2 prevented IL-1β-induced up-regulation of hepcidin. Our results clarify how commensal bacteria affect hepcidin expression and reveal a novel connection between IL-1β and activation of BMP signaling. They also suggest that there may be differences between mice and humans with respect to the mechanism by which IL-1β up-regulates hepcidin.

  5. Regulation of circadian gene expression in liver by systemic signals and hepatocyte oscillators.

    PubMed

    Kornmann, B; Schaad, O; Reinke, H; Saini, C; Schibler, U

    2007-01-01

    The mammalian circadian timing system has a hierarchical structure, in that a master pacemaker located in the suprachiasmatic nuclei (SCN) coordinates slave oscillators present in virtually all body cells. In both the SCN and peripheral organs, the rhythm-generating oscillators are self-sustained and cell-autonomous, and it is likely that the molecular makeup of master and slave oscillators is nearly identical. However, due to variations in period length, the phase coherence between peripheral oscillators in intact animals must be established by daily signals emanating directly or indirectly from the SCN master clock. The synchronization of individual cellular clocks in peripheral organs is probably accomplished by immediate-early genes that interpret the cyclic systemic signals and convey this phase information to core clock components. This model predicts that circadian gene expression in peripheral organs can be influenced either by systemic signals emanating from the SCN master clock, local oscillators, or both. We developed a transgenic mouse strain in which hepatocyte clocks are only operative when the tetracycline analog doxycycline is added to the food or drinking water. The genome-wide mapping of genes whose cyclic expression in liver does not depend on functional hepatocyte oscillators unveiled putative signaling pathways that may participate in the phase entrainment of peripheral clocks.

  6. Polarimetric Observation of Pulsars with Hexes

    NASA Astrophysics Data System (ADS)

    Xue, M.; Bhat, R.; Tremblay, S.; Ord, S.; Sobey, C.; Kirsten, F.

    2016-07-01

    The MWA VCS pipeline is now reliably generating high time resolution observations of radio pulsars in all four Stokes parameters. Here, we are proposing to test the polarimetric response of and our ability to calibrate the new Hex array currently under construction. These observation will provide data that will be used to study the pulsars themselves (including their emission mechanism and beam geometry), the interstellar medium and towards understanding the Galactic magnetic field. We are proposing a set of observations of three pulsars (J0034-0534, J0437-4715, and J2145-0750) at a wide range of hour angles to characterise the fidelity and stability of the polarimetric solutions with the hexes. The observation would be performed between 170-200 MHz and 140-170 MHz respectively. This project will form part of the PhD program of Mengyao Xue.

  7. 'HEXE' - X-ray observatory in space

    NASA Astrophysics Data System (ADS)

    1987-06-01

    An overview is given of the design concept and scientific goals of the High-Energy X-ray Experiment (HEXE), developed in the FRG (by the Max Planck Institute for Extraterrestrial Physics and the Astronomical Institute of Tuebingen University) for operation on the Soviet space station Mir. HEXE was launched to LEO using a Kvant vehicle on March 31, 1987; after initial docking problems, it was joined to Mir by two cosmonauts in a 3-hour EVA on April 12. HEXE has dimensions 45 x 45 x 75 cm and weight 180 kg; it employs an 800-sq-cm Tl-doped NaI/CsI phoswich detector for 15-250-keV X-rays, complementing the other Mir instruments: the ESTEC high-pressure gas-scintillation proportional counter (3-100 keV), the Soviet high-energy detector (20-800 keV), and the Dutch-British X-ray camera (2-30 keV). The Mir observations are intended to explore the energy spectra and time evolution of compact galactic and extragalactic objects.

  8. 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.

  9. Hepatocyte differentiation.

    PubMed

    Olsavsky Goyak, Katy M; Laurenzana, Elizabeth M; Omiecinski, Curtis J

    2010-01-01

    Increasingly, research suggests that for certain systems, animal models are insufficient for human toxicology testing. The development of robust, in vitro models of human toxicity is required to decrease our dependence on potentially misleading in vivo animal studies. A critical development in human toxicology testing is the use of human primary hepatocytes to model processes that occur in the intact liver. However, in order to serve as an appropriate model, primary hepatocytes must be maintained in such a way that they persist in their differentiated state. While many hepatocyte culture methods exist, the two-dimensional collagen "sandwich" system combined with a serum-free medium, supplemented with physiological glucocorticoid concentrations, appears to robustly maintain hepatocyte character. Studies in rat and human hepatocytes have shown that when cultured under these conditions, hepatocytes maintain many markers of differentiation including morphology, expression of plasma proteins, hepatic nuclear factors, phase I and II metabolic enzymes. Functionally, these culture conditions also preserve hepatic stress response pathways, such as the SAPK and MAPK pathways, as well as prototypical xenobiotic induction responses. This chapter will briefly review culture methodologies but will primarily focus on hallmark hepatocyte structural, expression and functional markers that characterize the differentiation status of the hepatocyte.

  10. Differential regulation of the mitogen-activated protein and stress-activated protein kinase cascades by adrenergic agonists in quiescent and regenerating adult rat hepatocytes.

    PubMed Central

    Spector, M S; Auer, K L; Jarvis, W D; Ishac, E J; Gao, B; Kunos, G; Dent, P

    1997-01-01

    To study the mechanisms by which catecholamines regulate hepatocyte proliferation after partial hepatectomy (PHX), hepatocytes were isolated from adult male rats 24 h after sham operation or two-thirds PHX and treated with catecholamines and other agonists. In freshly isolated sham cells, p42 mitogen-activated protein (MAP) kinase activity was stimulated by the alpha1-adrenergic agonist phenylephrine (PHE). Activation of p42 MAP kinase by growth factors was blunted by pretreatment of sham hepatocytes with glucagon but not by that with the beta2-adrenergic agonist isoproterenol (ISO). In PHX cells, the ability of PHE to activate p42 MAP kinase was dramatically reduced, whereas ISO became competent to inhibit p42 MAP kinase activation. PHE treatment of sham but not PHX and ISO treatment of PHX but not sham hepatocytes also activated the stress-activated protein (SAP) kinases p46/54 SAP kinase and p38 SAP kinase. These data demonstrate that an alpha1- to beta2-adrenergic receptor switch occurs upon PHX and results in an increase in SAP kinase versus MAP kinase signaling by catecholamines. In primary cultures of hepatocytes, ISO treatment of PHX but not sham cells inhibited [3H]thymidine incorporation. In contrast, PHE treatment of sham but not PHX cells stimulated [3H]thymidine incorporation, which was reduced by approximately 25 and approximately 95% with specific inhibitors of p42 MAP kinase and p38 SAP kinase function, respectively. Inhibition of the p38 SAP kinase also dramatically reduced basal [3H]thymidine incorporation. These data suggest that p38 SAP kinase plays a permissive role in liver regeneration. Alterations in the abilities of catecholamines to modulate the activities of protein kinase A and the MAP and SAP kinase pathways may represent one physiological mechanism by which these agonists can regulate hepatocyte proliferation after PHX. PMID:9199291

  11. Hepatocyte growth factor and transforming growth factor beta regulate 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression in rat hepatocyte primary cultures.

    PubMed Central

    Joaquin, M; Rosa, J L; Salvadó, C; López, S; Nakamura, T; Bartrons, R; Gil, J; Tauler, A

    1996-01-01

    Hepatocyte growth factor (HGF) and transforming growth factor beta (TGF-beta) are believed to be of major importance for hepatic regeneration after liver damage. We have studied the effect of these growth factors on fructose 2,6-bisphosphate (Fru-2,6-P2) levels and the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF2K/Fru-2,6-BPase) in rat hepatocyte primary cultures. Our results demonstrate that HGF activates the expression of the 6PF2K/Fru-2,6-BPase gene by increasing the levels of its mRNA. As a consequence of this activation, the amount of 6PF2K/Fru-2,6-BPase protein and 6-phosphofructo-2-kinase activity increased, which was reflected by a rise in Fru-2,6-P2 levels. In contrast, TGF-beta decreased the levels of 6PF2K/Fru-2,6-BPase mRNA, which led to a decrease in the amount of 6PF2K/Fru-2,6-BPase protein and Fru-2,6-P2. The different actions of HGF and TGF-beta on 6PF2K/Fru-2,6-BPase gene expression are concomitant with their effect on cell proliferation. Here we show that, in the absence of hormones, primary cultures of hepatocytes express the F-type isoenzyme. In addition, HGF increases the expression of this isoenzyme, and dexamethasone activates the L-type isoform. HGF and TGF-beta were able to inhibit this activation. PMID:8660288

  12. The role of lncRNA MALAT1 in the regulation of hepatocyte proliferation during liver regeneration

    PubMed Central

    Li, Cuicui; Chang, Lei; Chen, Zhiquan; Liu, Zhongzhong; Wang, Yanfeng; Ye, Qifa

    2017-01-01

    Exploring the biological functions of long non-coding RNAs (lncRNAs) has come to the foreground in recent years. Studies have indicated that the lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) not only regulates tumorigenesis in hepatocellular carcinoma, but also controls cell cycle progression in hematopoietic cells. The present study was designed to investigate the biological role of lncRNA MALAT1 in liver regeneration. We carried out a series of assays during liver regeneration following 2/3 partial hepatectomy in mice. We explored the functions of lncRNA MALAT1 with a series of functional analyses in vitro. We found that MALAT1 was upregulated during liver regeneration. Moreover, MALAT1 accelerated hepatocyte proliferation by stimulating cell cycle progression from the G1 to the S phase and inhibiting apoptosis in vitro. In addition, our findings also demonstrated that MALAT1 was regulated by p53 during liver regeneration, and that p53 may be a key upstream regulator of MALAT1 activity. Mechanistically, we found that MALAT1 activated the Wnt/β-catenin pathway by inhibiting the expression of Axin1 and adenomatous polyposis coli (APC), and subsequently promoting the expression of cyclin D1. On the whole, the findings of this study suggest that MALAT1 is a critical molecule for liver regeneration. Pharmacological interventions targeting MALAT1 may thus prove to be therapeutically beneficial in liver failure or liver transplantation by promoting liver regeneration. PMID:28075444

  13. Hepatocyte growth factor plays a dual role in regulating skeletal muscle satellite cell proliferation and differentiation.

    PubMed

    Gal-Levi, R; Leshem, Y; Aoki, S; Nakamura, T; Halevy, O

    1998-03-12

    The role of hepatocyte growth factor (HGF) and its receptor, c-met, in proliferation and differentiation of satellite cells was studied in primary cultures of chicken skeletal muscle satellite cells and a myogenic C2 cell line. HGF mRNA was expressed mainly in the myotubes of both cultures. The addition of conditioned medium derived from those cultures had a scattering effect on the canine kidney epithelial cell line, MDCK. In contrast, c-met mRNA levels decreased during cell differentiation of C2 and primary satellite cells. Application of exogenous HGF to chicken myoblasts resulted in their enhanced DNA synthesis. Among several growth factors, HGF was the first to induce DNA synthesis in quiescent satellite cells, thereby driving them into the cell cycle. Ectopic expression of chicken HGF in primary satellite cells suppressed the activation of muscle-regulatory gene reporter constructs MCK-CAT, MRF4-CAT, MEF2-CAT and 4Rtk-CAT, as well as the gene expression of MyoD and myogenin, and MHC protein expression. Ectopic MyoD reversed HGF's inhibitory effect on MCK transactivation. These data suggest that HGF inhibits cell differentiation by inhibiting the activity of basic helix-loop-helix (bHLH)/E protein heterodimers, thus inhibiting myogenic determination factor activity and subsequent muscle-specific protein expression. During muscle growth and regeneration, HGF plays a dual role in satellite-cell myogenesis, affecting both the proliferation and differentiation of these cells in a paracrine fashion.

  14. Regulation of amino acid transport in isolated rat hepatocytes during development

    SciTech Connect

    Leoni, S.; Spagnuolo, S.; Dini, L.; Devirgiliis, L.C.

    1987-01-01

    The effect of amino acid depletion or supplementation and the effect of glucagon and insulin on the amino acid transport mediated by system A were investigated by determining the uptake of either 2-amino (1-/sup 14/C)isobutyric acid (AIB) or N-methyl 2-amino (1-/sup 14/C)isobutyric acid (MeAIB) in rat hepatocytes, freshly isolated at different stages of pre- and postnatal development. The data obtained show that the Na/sup +/ -dependent uptake was higher at the earliest developmental stages, and steadily decreased until the adult level. The hormones increased AIB and MeAIB uptake enhancing the V/sub max/, while the K/sub m/ was unchanged. This effect was evident in cells from adult and 18-20-day-old fetuses, while no response was present before the 18th day of fetal life and in the prenatal period. Actinomycin D or cycloheximide abolished this hormone-dependent increase. A decrease in AIB and MeAIB transport after incubation in an amino acid-rich medium was demonstrated at all ages tested, but was particularly evident in the prenatal life. The increase in the activity of the system following amino acid starvation was shown to be mostly dependent from de novo protein synthesis in the fetal life; on the contrary in the adult the increase appeared to be more linked to the release from transinhibition of the transport.

  15. 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

  16. 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

  17. Intracellular degradation in the regulation of secretion of apolipoprotein B-100 by rabbit hepatocytes.

    PubMed

    Cartwright, I J; Higgins, J A

    1996-03-15

    Isolated rabbit hepatocytes were incubated with [35S]methionine to label intracellular pools of apolipoprotein B (apo-B). The cells were then reincubated with an excess of unlabelled methionine in the presence of oleate or protease inhibitors and the intracellular sites of accumulation of radiolabelled apo-B and the mass of apo-B were determined by isolation and analysis of subcellular fractions. Oleate or inhibitors of metalloproteases (o-phenanthroline), serine proteases (aprotinin), serine/cysteine proteases (leupeptin) or cysteins proteases (calpain inhibitor I; ALLN) but not aspartate proteases (pepstatin) resulted in inhibition of the cellular degradation of apo-B. The effect of o-phenanthroline was reversed by the addition of zinc ions. Oleate, o-phenanthroline and leupeptin also stimulated secretion of radiolabelled apo-B; the effects of the inhibitors and oleate were additive, suggesting that they could act via different mechanisms. o-Phenanthroline caused accumulation of apo-B in the rough endoplasmic reticulum (RER) and smooth endoplasmic reticulum (SER) membranes; leupeptin caused accumulation of apo-B in the SER and cis-Golgi membranes, and ALLN and aprotinin caused accumulation of apo-B in the trans-Golgi membranes. These results suggest that intracellular degradation of apo-B occurs in the endoplasmic reticulum and in the trans-Golgi membranes and involves different proteases. Apo-B that accumulates in the ER membrane can be diverted into the lumen for secretion; however, apo-B that accumulates in the trans-Golgi membrane is irretrievably diverted from secretion.

  18. Hepatocyte Nuclear Factor 4α Controls Iron Metabolism and Regulates Transferrin Receptor 2 in Mouse Liver*

    PubMed Central

    Matsuo, Shunsuke; Ogawa, Masayuki; Muckenthaler, Martina U.; Mizui, Yumiko; Sasaki, Shota; Fujimura, Takafumi; Takizawa, Masayuki; Ariga, Nagayuki; Ozaki, Hiroaki; Sakaguchi, Masakiyo; Gonzalez, Frank J.; Inoue, Yusuke

    2015-01-01

    Iron is an essential element in biological systems, but excess iron promotes the formation of reactive oxygen species, resulting in cellular toxicity. Several iron-related genes are highly expressed in the liver, a tissue in which hepatocyte nuclear factor 4α (HNF4α) plays a critical role in controlling gene expression. Therefore, the role of hepatic HNF4α in iron homeostasis was examined using liver-specific HNF4α-null mice (Hnf4aΔH mice). Hnf4aΔH mice exhibit hypoferremia and a significant change in hepatic gene expression. Notably, the expression of transferrin receptor 2 (Tfr2) mRNA was markedly decreased in Hnf4aΔH mice. Promoter analysis of the Tfr2 gene showed that the basal promoter was located at a GC-rich region upstream of the transcription start site, a region that can be transactivated in an HNF4α-independent manner. HNF4α-dependent expression of Tfr2 was mediated by a proximal promoter containing two HNF4α-binding sites located between the transcription start site and the translation start site. Both the GC-rich region of the basal promoter and the HNF4α-binding sites were required for maximal transactivation. Moreover, siRNA knockdown of HNF4α suppressed TFR2 expression in human HCC cells. These results suggest that Tfr2 is a novel target gene for HNF4α, and hepatic HNF4α plays a critical role in iron homeostasis. PMID:26527688

  19. Human CB1 Receptor Isoforms, present in Hepatocytes and β-cells, are Involved in Regulating Metabolism

    PubMed Central

    González-Mariscal, Isabel; Krzysik-Walker, Susan M.; Doyle, Máire E.; Liu, Qing-Rong; Cimbro, Raffaello; Santa-Cruz Calvo, Sara; Ghosh, Soumita; Cieśla, Łukasz; Moaddel, Ruin; Carlson, Olga D.; Witek, Rafal P.; O’Connell, Jennifer F.; Egan, Josephine M.

    2016-01-01

    Therapeutics aimed at blocking the cannabinoid 1 (CB1) receptor for treatment of obesity resulted in significant improvements in liver function, glucose uptake and pancreatic β-cell function independent of weight loss or CB1 receptor blockade in the brain, suggesting that peripherally-acting only CB1 receptor blockers may be useful therapeutic agents. Neuropsychiatric side effects and lack of tissue specificity precluded clinical use of first-generation, centrally acting CB1 receptor blockers. In this study we specifically analyzed the potential relevance to diabetes of human CB1 receptor isoforms in extraneural tissues involved in glucose metabolism. We identified an isoform of the human CB1 receptor (CB1b) that is highly expressed in β-cells and hepatocytes but not in the brain. Importantly, CB1b shows stronger affinity for the inverse agonist JD-5037 than for rimonabant compared to CB1 full length. Most relevant to the field, CB1b is a potent regulator of adenylyl cyclase activity in peripheral metabolic tissues. CB1b blockade by JD-5037 results in stronger adenylyl cyclase activation compared to rimonabant and it is a better enhancer of insulin secretion in β-cells. We propose this isoform as a principal pharmacological target for the treatment of metabolic disorders involving glucose metabolism. PMID:27641999

  20. Human CB1 Receptor Isoforms, present in Hepatocytes and β-cells, are Involved in Regulating Metabolism.

    PubMed

    González-Mariscal, Isabel; Krzysik-Walker, Susan M; Doyle, Máire E; Liu, Qing-Rong; Cimbro, Raffaello; Santa-Cruz Calvo, Sara; Ghosh, Soumita; Cieśla, Łukasz; Moaddel, Ruin; Carlson, Olga D; Witek, Rafal P; O'Connell, Jennifer F; Egan, Josephine M

    2016-09-19

    Therapeutics aimed at blocking the cannabinoid 1 (CB1) receptor for treatment of obesity resulted in significant improvements in liver function, glucose uptake and pancreatic β-cell function independent of weight loss or CB1 receptor blockade in the brain, suggesting that peripherally-acting only CB1 receptor blockers may be useful therapeutic agents. Neuropsychiatric side effects and lack of tissue specificity precluded clinical use of first-generation, centrally acting CB1 receptor blockers. In this study we specifically analyzed the potential relevance to diabetes of human CB1 receptor isoforms in extraneural tissues involved in glucose metabolism. We identified an isoform of the human CB1 receptor (CB1b) that is highly expressed in β-cells and hepatocytes but not in the brain. Importantly, CB1b shows stronger affinity for the inverse agonist JD-5037 than for rimonabant compared to CB1 full length. Most relevant to the field, CB1b is a potent regulator of adenylyl cyclase activity in peripheral metabolic tissues. CB1b blockade by JD-5037 results in stronger adenylyl cyclase activation compared to rimonabant and it is a better enhancer of insulin secretion in β-cells. We propose this isoform as a principal pharmacological target for the treatment of metabolic disorders involving glucose metabolism.

  1. Kinetics of myc-max-mad gene expression during hepatocyte proliferation in vivo: Differential regulation of mad family and stress-mediated induction of c-myc.

    PubMed

    Mauleon, Itsaso; Lombard, Marie-Noëlle; Muñoz-Alonso, Maria J; Cañelles, Matilde; Leon, Javier

    2004-02-01

    Mad proteins (Mad1, Mxi1, Mad3, Mad4, Mnt/Rox) are biochemical and biological antagonists of c-Myc oncoprotein. Mad-Max dimers repress the transcription of the same target genes activated by Myc-Max dimers. Despite the critical role of Max and Mad proteins as modulators of c-Myc functions, there are no comparative data on their regulation in vivo. We carried out a systematic analysis of c-myc, max, and mad family expression in a model of synchronized cell proliferation in vivo in adult tissues, that is, rat hepatocytes after partial hepatectomy. We confirmed the previously reported early peak of c-myc expression after hepatectomy but we show that it did not correlate with hepatocyte proliferation as it also occurred in sham-operated animals as a result of surgical stresses. A second peak of c-myc expression was observed later, at the time of the wave of DNA synthesis. No such expression was detected in sham-operated rat quiescent hepatocytes. max expression increased around 4-16 h after hepatectomy, before the peaks of c-myc and DNA synthesis. mxi1 and mad4 were slightly downregulated during liver regeneration. mnt/rox expression did not change. These expression patterns suggest a role of Myc-Max for efficient mitogenic response of hepatocytes. We also analyzed the effects of Myc and Max ectopic expression on the clonogenic growth of the rat hepatoma cells. Expression of c-Myc and Max increased clonogenic growth, whereas the reduction of c-Myc levels by an antisense vector decreased growth. The results suggest nonredundant roles for mad genes in hepatocyte proliferation and point to c-Myc as a putative target for anticancer therapy of liver cancer.

  2. Hepatocyte Polarity

    PubMed Central

    Treyer, Aleksandr; Müsch, Anne

    2013-01-01

    Hepatocytes, like other epithelia, are situated at the interface between the organism’s exterior and the underlying internal milieu and organize the vectorial exchange of macromolecules between these two spaces. To mediate this function, epithelial cells, including hepatocytes, are polarized with distinct luminal domains that are separated by tight junctions from lateral domains engaged in cell-cell adhesion and from basal domains that interact with the underlying extracellular matrix. Despite these universal principles, hepatocytes distinguish themselves from other nonstriated epithelia by their multipolar organization. Each hepatocyte participates in multiple, narrow lumina, the bile canaliculi, and has multiple basal surfaces that face the endothelial lining. Hepatocytes also differ in the mechanism of luminal protein trafficking from other epithelia studied. They lack polarized protein secretion to the luminal domain and target single-spanning and glycosylphosphatidylinositol-anchored bile canalicular membrane proteins via transcytosis from the basolateral domain. We compare this unique hepatic polarity phenotype with that of the more common columnar epithelial organization and review our current knowledge of the signaling mechanisms and the organization of polarized protein trafficking that govern the establishment and maintenance of hepatic polarity. The serine/threonine kinase LKB1, which is activated by the bile acid taurocholate and, in turn, activates adenosine monophosphate kinase-related kinases including AMPK1/2 and Par1 paralogues has emerged as a key determinant of hepatic polarity. We propose that the absence of a hepatocyte basal lamina and differences in cell-cell adhesion signaling that determine the positioning of tight junctions are two crucial determinants for the distinct hepatic and columnar polarity phenotypes. PMID:23720287

  3. 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.

  4. Extracellular Signal-Regulated Kinase Is an Endogenous Signal Retaining the Nuclear Constitutive Active/Androstane Receptor (CAR) in the Cytoplasm of Mouse Primary Hepatocytes

    PubMed Central

    Koike, Chika; Moore, Rick; Negishi, Masahiko

    2007-01-01

    The nuclear receptor constitutive active/androstane receptor (CAR) is sequestered in the cytoplasm of liver cells before its activation by therapeutic drugs and xenobiotics such as phenobarbital (PB) and 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) in mouse liver, the regulatory mechanism of which remains poorly understood. Given the finding that epidermal growth factor repressed PB activation of CAR-mediated transcription (Mol Pharmacol 65:172–180, 2004), here we investigated the regulatory role of hepatocyte growth factor (HGF)-mediated signal in sequestering CAR in the cytoplasm of mouse primary hepatocytes. HGF treatment effectively repressed the induction of endogenous CYP2b10 gene by PB and TCPOBOP in mouse primary hepatocytes. On the other hand, inhibition by 1,4-diamino-2,3-dicyano-1,4-bis(methyl-thio)butadiene (U0126) of an HGF downstream kinase mitogen-activated protein kinase kinase (MEK) induced the Cyp2b10 gene and up-regulated the CAR-regulated promoter activity in the absence of TCPOBOP. HGF treatment increased phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 in the cytosol, thus decreasing the TCPOBOP-induced nuclear accumulation of CAR. In contrast, U0126 dephosphorylated ERK1/2 and increased nuclear CAR accumulation in the absence of TCPOBOP. These results are consistent with the conclusion that the HGF-dependent phosphorylation of ERK1/2 is the endogenous signal that sequesters CAR in the cytoplasm of mouse primary hepatocytes. PMID:17314319

  5. Metabolic hormones regulate basal and growth hormone-dependent igf2 mRNA level in primary cultured coho salmon hepatocytes: effects of insulin, glucagon, dexamethasone, and triiodothyronine.

    PubMed

    Pierce, A L; Dickey, J T; Felli, L; Swanson, P; Dickhoff, W W

    2010-03-01

    Igf1 and Igf2 stimulate growth and development of vertebrates. Circulating Igfs are produced by the liver. In mammals, Igf1 mediates the postnatal growth-promoting effects of growth hormone (Gh), whereas Igf2 stimulates fetal and placental growth. Hepatic Igf2 production is not regulated by Gh in mammals. Little is known about the regulation of hepatic Igf2 production in nonmammalian vertebrates. We examined the regulation of igf2 mRNA level by metabolic hormones in primary cultured coho salmon hepatocytes. Gh, insulin, the glucocorticoid agonist dexamethasone (Dex), and glucagon increased igf2 mRNA levels, whereas triiodothyronine (T(3)) decreased igf2 mRNA levels. Gh stimulated igf2 mRNA at physiological concentrations (0.25x10(-9) M and above). Insulin strongly enhanced Gh stimulation of igf2 at low physiological concentrations (10(-11) M and above), and increased basal igf2 (10(-8) M and above). Dex stimulated basal igf2 at concentrations comparable to those of stressed circulating cortisol (10(-8) M and above). Glucagon stimulated basal and Gh-stimulated igf2 at supraphysiological concentrations (10(-7) M and above), whereas T(3) suppressed basal and Gh-stimulated igf2 at the single concentration tested (10(-7) M). These results show that igf2 mRNA level is highly regulated in salmon hepatocytes, suggesting that liver-derived Igf2 plays a significant role in salmon growth physiology. The synergistic regulation of igf2 by insulin and Gh in salmon hepatocytes is similar to the regulation of hepatic Igf1 production in mammals.

  6. Proanthocyanidin Protects Human Embryo Hepatocytes from Fluoride-Induced Oxidative Stress by Regulating Iron Metabolism.

    PubMed

    Niu, Qiang; Mu, Lati; Li, Shugang; Xu, Shangzhi; Ma, Ruling; Guo, Shuxia

    2016-02-01

    To investigate whether grape seed proanthocyanidin extract (GSPE) antagonizes fluoride-induced oxidative injury by regulating iron metabolism, human embryo hepatic cells (L-02) were incubated with sodium fluoride (NaF, 80 mg/L) and/or GSPE (100 μmol/L) for 24 h. Results showed the glutathione peroxidase (GSH-Px) content, superoxide dismutase (SOD) activity, and total antioxidant capacity (T-AOC) level of the NaF group were significantly lower than that of the control group (P < 0.05), while malondialdehyde (MDA) content increased in the NaF group compared with the control group (P < 0.05). Moreover, the indexes mentioned above showed opposite changes in the NaF + GSPE group. In addition, iron content significantly increased in the NaF group compared to the control group(P < 0.05) and significantly decreased in the NaF + GSPE group compared to the NaF group (P < 0.05). Furthermore, hepcidin (coded by HAMP) messenger RNA (mRNA) expression significantly increased in the NaF group compared to the control group(P < 0.05) and significantly decreased in the NaF + GSPE group compared to the NaF group (P < 0.05). Ferroportin 1 (coded by FPN1) mRNA expression significantly decreased in the NaF group compared to the control group (P < 0.05) and significantly increased in the NaF + GSPE group compared to the NaF group (P < 0.05). These results indicate that GSPE provides significant cellular protection against oxidative stress induced by excessive fluoride via the iron metabolism regulation.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. Estrogen regulates hepcidin expression via GPR30-BMP6-dependent signaling in hepatocytes.

    PubMed

    Ikeda, Yasumasa; Tajima, Soichiro; Izawa-Ishizawa, Yuki; Kihira, Yoshitaka; Ishizawa, Keisuke; Tomita, Shuhei; Tsuchiya, Koichiro; Tamaki, Toshiaki

    2012-01-01

    Hepcidin, a liver-derived iron regulatory protein, plays a crucial role in iron metabolism. It is known that gender differences exist with respect to iron storage in the body; however, the effects of sex steroid hormones on iron metabolism are not completely understood. We focused on the effects of the female sex hormone estrogen on hepcidin expression. First, ovariectomized (OVX) and sham-operated mice were employed to investigate the effects of estrogen on hepcidin expression in an in vivo study. Hepcidin expression was decreased in the livers of OVX mice compared to the sham-operated mice. In OVX mice, bone morphologic protein-6 (BMP6), a regulator of hepcidin, was also found to be downregulated in the liver, whereas ferroportin (FPN), an iron export protein, was upregulated in the duodenum. Both serum and liver iron concentrations were elevated in OVX mice relative to their concentrations in sham-operated mice. In in vitro studies, 17β-estradiol (E(2)) increased the mRNA expression of hepcidin in HepG2 cells in a concentration-dependent manner. E(2)-induced hepatic hepcidin upregulation was not inhibited by ICI 182720, an inhibitor of the estrogen receptor; instead, hepcidin expression was increased by ICI 182720. E(2) and ICI 182720 exhibit agonist actions with G-protein coupled receptor 30 (GPR30), the 7-transmembrane estrogen receptor. G1, a GPR30 agonist, upregulated hepcidin expression, and GPR30 siRNA treatment abolished E(2)-induced hepcidin expression. BMP6 expression induced by E(2) was abolished by GPR30 silencing. Finally, both E(2) and G1 supplementation restored reduced hepatic hepcidin and BMP6 expression and reversed the augmentation of duodenal FPN expression in the OVX mice. In contrast, serum hepcidin was elevated in OVX mice, which was reversed in these mice with E(2) and G1. Thus, estrogen is involved in hepcidin expression via a GPR30-BMP6-dependent mechanism, providing new insight into the role of estrogen in iron metabolism.

  13. 1. GENERAL VIEW. NOTE HEX SIGNS (BLACK AND WHITE COLORS), ...

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

    1. GENERAL VIEW. NOTE HEX SIGNS (BLACK AND WHITE COLORS), WINDOW AND DOOR 'HEADS', AND SCALLOP PATTERN (ON BOTTOM OF OVERHANG). - Decorated Red Barn, State Route 309 vicinity, Center Valley, Lehigh County, PA

  14. Common HEXB polymorphisms reduce serum HexA and HexB enzymatic activities, potentially masking Tay-Sachs disease carrier identification.

    PubMed

    Vallance, Hilary; Morris, Tara J; Coulter-Mackie, Marion; Lim-Steele, Joyce; Kaback, Michael

    2006-02-01

    A DNA-proven Tay-Sachs disease (TSD) carrier and his brother were found to have serum percent Hexosaminidase A (%HexA) enzymatic activities in the non-carrier range, while the leukocyte %HexA profiles clearly identified them as TSD heterozygotes. Both their serum HexA and HexB enzymatic activities were below reference range, suggesting inheritance of mutations in both the HEXA (alpha-subunit) and HEXB (beta-subunit) genes. DNA sequencing revealed that both individuals, carried the common HEXA 1277_1278insTATC mutation, and two common HEXB polymorphisms: [619A>G (+) delTG]. To determine if these HEXB polymorphisms reduce HexA and HexB enzymatic activities, 69 DNA samples from subjects previously screened enzymatically in both serum and leukocytes for TSD carrier status were selected for either high, mid-range or low serum Total Hex (defined as the sum of HexA and HexB) activities and were tested for the HEXB mutations. Further, three additional TSD carriers ascertained by the atypical pattern of normal serum %HexA but carrier leukocyte %HexA, were found to have the [delTG (+) 619A>G] genotype. In addition, the frequency of the [delTG (+) 619A>G] genotype was significantly higher (P < 0.01) in subjects with low serum HexB enzymatic activities. Given the high frequency of the [delTG (+) 619A>G] haplotype in the Ashkenazi Jewish population (approximately 10%), up to 10% of TSD carriers may have normal serum %HexA values with low total Hex. Accordingly, serum %HexA should not be the sole criterion used for carrier status determination. Where total Hex activity is reduced, further testing with leukocyte Hex profiles is indicated.

  15. 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.

  16. Reciprocal regulation of farnesoid X receptor α activity and hepatitis B virus replication in differentiated HepaRG cells and primary human hepatocytes.

    PubMed

    Radreau, Pauline; Porcherot, Marine; Ramière, Christophe; Mouzannar, Karim; Lotteau, Vincent; André, Patrice

    2016-09-01

    Hepatitis B virus (HBV) and bile salt metabolism seem tightly connected. HBV enters hepatocytes by binding to sodium taurocholate cotransporting polypeptide (NTCP), the genome of which contains 2 active farnesoid X receptor (FXR) α response elements that participate in HBV transcriptional activity. We investigated in differentiated HepaRG cells and in primary human hepatocytes (PHHs) effects of FXR activation on HBV replication and of infection on the FXR pathway. In HepaRG cells, FXR agonists (6-ethyl chenodeoxycholic acid and GW4064), but no antagonist, and an FXR-unrelated bile salt inhibited viral mRNA, DNA, and protein production (IC50, 0.1-0.5 μM) and reduced covalently closed circular DNA pool size. These effects were independent of the NTCP inhibitor cyclosporine-A, which suggests inhibition occurred at a postentry step. Similar results were obtained in PHHs with GW4064. Infection of these cells increased expression of FXR and modified expression of FXR-regulated genes SHP, APOA1, NTCP, CYP7A1, and CYP8B1 with a more pronounced effect in PHHs than in HepaRG cells. FXR agonists reversed all but one of the HBV-induced FXR gene profile modifications. HBV replication and FXR regulation seem to be interdependent, and altered bile salt metabolism homeostasis might contribute to the persistence of HBV infection.-Radreau, P., Porcherot, M., Ramière, C., Mouzannar, K., Lotteau, V., André, P. Reciprocal regulation of farnesoid X receptor α activity and hepatitis B virus replication in differentiated HepaRG cells and primary human hepatocytes.

  17. 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.

  18. Current status of hepatocyte xenotransplantation.

    PubMed

    Meier, Raphael P H; Navarro-Alvarez, Nalu; Morel, Philippe; Schuurman, Henk-Jan; Strom, Stephen; Bühler, Leo H

    2015-11-01

    The treatment of acute liver failure, a condition with high mortality, comprises optimal clinical care, and in severe cases liver transplantation. However, there are limitations in availability of organ donors. Hepatocyte transplantation is a promising alternative that could fill the medical need, in particular as the bridge to liver transplantation. Encapsulated porcine hepatocytes represent an unlimited source that could function as a bioreactor requiring minimal immunosuppression. Besides patients with acute liver failure, patients with alcoholic hepatitis who are unresponsive to a short course of corticosteroids are a target for hepatocyte transplantation. In this review we present an overview of the innate immune barriers in hepatocyte xenotransplantation, including the role of complement and natural antibodies; the role of phagocytic cells and ligands like CD47 in the regulation of phagocytic cells; and the role of Natural Killer cells. We present also some illustrations of physiological species incompatibilities in hepatocyte xenotransplantation, such as incompatibilities in the coagulation system. An overview of the methodology for cell microencapsulation is presented, followed by proof-of-concept studies in rodent and nonhuman primate models of fulminant liver failure: these studies document the efficacy of microencapsulated porcine hepatocytes which warrants progress towards clinical application. Lastly, we present an outline of a provisional clinical trial, that upon completion of preclinical work could start within the upcoming 2-3 years.

  19. 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

  20. The High Energy X-ray Probe (HEX-P)

    NASA Astrophysics Data System (ADS)

    Harrison, Fiona; HEX-P Collaboration Collaboration

    2017-01-01

    The High-Energy X-ray Probe (HEX-P) is a probe-class ( 500M) next-generation high-energy X-ray observatory with broadband (2-200 keV) response and 40 times the sensitivity of any previous mis-sion in the 10-80 keV band, and >500 times the sensitivity of any previous mission in the 80-200 keV band. Intended to launch contemporaneously with Athena, HEX-P will provide fundamental new discoveries that range from resolving 90% of the X-ray background at its peak, to measuring the cosmic evolution of black hole spin, to studying faint X-ray populations in nearby galaxies. Based on NuSTAR heritage, HEX-P requires only modest technology development, and could easily be executed within the next decade.

  1. Curcumin regulates the metabolism of low density lipoproteins by improving the C-to-U RNA editing efficiency of apolipoprotein B in primary rat hepatocytes.

    PubMed

    Tian, Nan; Li, Xiaoling; Luo, Yang; Han, Ziwu; Li, Zhaohui; Fan, Chunlei

    2014-01-01

    There are two isoforms of apolipoprotein B (apoB) in mammals: apoB-100 and apoB-48. The latter is generated by C-to-U editing of apoB mRNA, catalyzed by the apolipoprotein B mRNA editing enzyme, namely, catalytic polypeptide 1 (APOBEC-1). Lipid particles containing apoB-48 are cleared from the plasma more rapidly than those containing apoB-100 and thus do not contribute to plaque formation in the arterial wall. In the present study, we analyzed whether curcumin is capable of regulating lipid metabolism by improving the level of apoB mRNA editing. The cytotoxicity of curcumin in hepatocytes was determined using the 3-(4,5-dimethylthiazol‑2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the levels of APOBEC-1 mRNA and protein were analyzed by real-time quantitative polymerase chain reaction (qRT-PCR) and western blotting. The efficiency of apoB mRNA editing was determined by reverse transcription PCR (RT-PCR) products and cloning sequencing analysis. We demonstrated that curcumin concentrations up to 70 µM had no significant cytotoxic effects on primary rat hepatocytes at 24 h. At 15 µM, curcumin significantly increased the expression of APOBEC-1 mRNA and protein, and increased the editing level of apoB mRNA from 3.13 to 7.53%. At 25 µM, curcumin reduced the expression of APOBEC-1; however, it did not affect the apoB mRNA editing level. Our data suggested that curcumin at a concentration of 15 µM raised the level of apoB-48 and reduced the level of apoB-100 by increasing the expression of APOBEC-1 in primary rat hepatocytes; therefore, curcumin may be a novel preventative therapy for atherosclerosis.

  2. Hepatic Tissue Environment in NEMO-Deficient Mice Critically Regulates Positive Selection of Donor Cells after Hepatocyte Transplantation

    PubMed Central

    Kaldenbach, Michaela; Cubero, Francisco Javier; Erschfeld, Stephanie; Liedtke, Christian; Trautwein, Christian; Streetz, Konrad

    2014-01-01

    Background Hepatocyte transplantation (HT) is a promising alternative treatment strategy for end-stage liver diseases compared with orthotopic liver transplantation. A limitation for this approach is the low engraftment of donor cells. The deletion of the I-kappa B kinase-regulatory subunit IKKγ/NEMO in hepatocytes prevents nuclear factor (NF)-kB activation and triggers spontaneous liver apoptosis, chronic hepatitis and the development of liver fibrosis and hepatocellular carcinoma. We hypothesized that NEMOΔhepa mice may therefore serve as an experimental model to study HT. Methods Pre-conditioned NEMOΔhepa mice were transplanted with donor-hepatocytes from wildtype (WT) and mice deficient for the pro-apoptotic mediator Caspase-8 (Casp8Δhepa). Results Transplantation of isolated WT-hepatocytes into pre-conditioned NEMOΔhepa mice resulted in a 6-7 fold increase of donor cells 12 weeks after HT, while WT-recipients showed no liver repopulation. The use of apoptosis-resistant Casp8Δhepa-derived donor cells further enhanced the selection 3-fold after 12-weeks and up to 10-fold increase after 52 weeks compared with WT donors. While analysis of NEMOΔhepa mice revealed strong liver injury, HT-recipient NEMOΔhepa mice showed improved liver morphology and decrease in serum transaminases. Concomitant with these findings, the histological examination elicited an improved liver tissue architecture associated with significantly lower levels of apoptosis, decreased proliferation and a lesser amount of liver fibrogenesis. Altogether, our data clearly support the therapeutic benefit of the HT procedure into NEMOΔhepa mice. Conclusion This study demonstrates the feasibility of the NEMOΔhepa mouse as an in vivo tool to study liver repopulation after HT. The improvement of the characteristic phenotype of chronic liver injury in NEMOΔhepa mice after HT suggests the therapeutic potential of HT in liver diseases with a chronic inflammatory phenotype and opens a new door for

  3. 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.

  4. 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

  5. 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

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

    PubMed

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

    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.

  7. 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

  8. Human circulating monocytes internalize 125I-insulin in a similar fashion to rat hepatocytes: relevance to receptor regulation in target and nontarget tissues.

    PubMed

    Grunberger, G; Robert, A; Carpentier, J L; Dayer, J M; Roth, A; Stevenson, H C; Orci, L; Gorden, P

    1985-08-01

    Circulating monocytes bind 125I-insulin in a specific fashion and have been used to analyze the ambient receptor status in humans. When freshly isolated circulating monocytes are incubated with 125I-insulin and examined by electron microscopic autoradiography, approximately 18% of the labeled material is internalized after 15 minutes at 37 degrees C. By 2 hours at 37 degrees C, approximately one half of the 125I-insulin is internalized. Internalization occurs also at 15 degrees C but at a slower rate. Furthermore, the monocytes bind and internalize 125I-insulin in a manner that mirrors that of major target tissues, such as rat hepatocytes. These data suggest that the insulin receptor of the circulating monocyte might be regulated by adsorptive endocytosis in a manner analogous to that of target tissue, such as the liver.

  9. Extra virgin olive oil phenols down-regulate lipid synthesis in primary-cultured rat-hepatocytes.

    PubMed

    Priore, Paola; Siculella, Luisa; Gnoni, Gabriele Vincenzo

    2014-07-01

    Hydroxytyrosol, tyrosol, and oleuropein, the main phenols present in extra virgin olive oil, have been reported to exert several biochemical and pharmacological effects. Here, we investigated the short-term effects of these compounds on lipid synthesis in primary-cultured rat-liver cells. Hydroxytyrosol, tyrosol and oleuropein inhibited both de novo fatty acid and cholesterol syntheses without an effect on cell viability. The inhibitory effect of individual compounds was already evident within 2 h of 25 μM phenol addition to the hepatocytes. The degree of cholesterogenesis reduction was similar for all phenol treatments (-25/30%), while fatty acid synthesis showed the following order of inhibition: hydroxytyrosol (-49%) = oleuropein (-48%) > tyrosol (-30%). A phenol-induced reduction of triglyceride synthesis was also detected. To clarify the lipid-lowering mechanism of these compounds, their influence on the activity of key enzymes of fatty acid biosynthesis (acetyl-CoA carboxylase and fatty acid synthase), triglyceride synthesis (diacylglycerol acyltransferase) and cholesterogenesis (3-hydroxy-3-methyl-glutaryl-CoA reductase) was investigated in situ by using digitonin-permeabilized hepatocytes. Acetyl-CoA carboxylase, diacylglycerol acyltransferase and 3-hydroxy-3-methyl-glutaryl-CoA reductase activities were reduced after 2 h of 25 μM phenol treatment. No change in fatty acid synthase activity was observed. Acetyl-CoA carboxylase inhibition (hydroxytyrosol, -41%, = oleuropein, -38%, > tyrosol, -17%) appears to be mediated by phosphorylation of AMP-activated protein kinase. These findings suggest that a decrease in hepatic lipid synthesis may represent a potential mechanism underlying the reported hypolipidemic effect of phenols of extra virgin olive oil.

  10. 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.

  11. An anti-PCSK9 antibody reduces LDL-cholesterol on top of a statin and suppresses hepatocyte SREBP-regulated genes.

    PubMed

    Zhang, Liwen; McCabe, Timothy; Condra, Jon H; Ni, Yan G; Peterson, Laurence B; Wang, Weirong; Strack, Alison M; Wang, Fubao; Pandit, Shilpa; Hammond, Holly; Wood, Dana; Lewis, Dale; Rosa, Ray; Mendoza, Vivienne; Cumiskey, Anne Marie; Johns, Douglas G; Hansen, Barbara C; Shen, Xun; Geoghagen, Neil; Jensen, Kristian; Zhu, Lei; Wietecha, Karol; Wisniewski, Douglas; Huang, Lingyi; Zhao, Jing Zhang; Ernst, Robin; Hampton, Richard; Haytko, Peter; Ansbro, Frances; Chilewski, Shannon; Chin, Jayne; Mitnaul, Lyndon J; Pellacani, Andrea; Sparrow, Carl P; An, Zhiqiang; Strohl, William; Hubbard, Brian; Plump, Andrew S; Blom, Daniel; Sitlani, Ayesha

    2012-01-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a promising therapeutic target for treating coronary heart disease. We report a novel antibody 1B20 that binds to PCSK9 with sub-nanomolar affinity and antagonizes PCSK9 function in-vitro. In CETP/LDLR-hemi mice two successive doses of 1B20, administered 14 days apart at 3 or 10 mpk, induced dose dependent reductions in LDL-cholesterol (≥ 25% for 7-14 days) that correlated well with the extent of PCSK9 occupancy by the antibody. In addition, 1B20 induces increases in total plasma antibody-bound PCSK9 levels and decreases in liver mRNA levels of SREBP-regulated genes PCSK9 and LDLR, with a time course that parallels decreases in plasma LDL-cholesterol (LDL-C). Consistent with this observation in mice, in statin-responsive human primary hepatocytes, 1B20 lowers PCSK9 and LDLR mRNA levels and raises serum steady-state levels of antibody-bound PCSK9. In addition, mRNA levels of several SREBP regulated genes involved in cholesterol and fatty-acid synthesis including ACSS2, FDPS, IDI1, MVD, HMGCR, and CYP51A1 were decreased significantly with antibody treatment of primary human hepatocytes. In rhesus monkeys, subcutaneous (SC) dosing of 1B20 dose-dependently induces robust LDL-C lowering (maximal ~70%), which is correlated with increases in target engagement and total antibody-bound PCSK9 levels. Importantly, a combination of 1B20 and Simvastatin in dyslipidemic rhesus monkeys reduced LDL-C more than either agent alone, consistent with a mechanism of action that predicts additive effects of anti-PCSK9 agents with statins. Our results suggest that antibodies targeting PCSK9 could provide patients powerful LDL lowering efficacy on top of statins, and lower cardiovascular risk.

  12. 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.

  13. β-Adrenergic agonist and antagonist regulation of autophagy in HepG2 cells, primary mouse hepatocytes, and mouse liver.

    PubMed

    Farah, Benjamin L; Sinha, Rohit A; Wu, Yajun; Singh, Brijesh K; Zhou, Jin; Bay, Boon-Huat; Yen, Paul M

    2014-01-01

    Autophagy recently has been shown to be involved in normal hepatic function and in pathological conditions such as non-alcoholic fatty liver disease. Adrenergic signalling also is an important regulator of hepatic metabolism and function. However, currently little is known about the potential role of adrenergic signaling on hepatic autophagy, and whether the β-adrenergic receptor itself may be a key regulator of autophagy. To address these issues, we investigated the actions of the β2-adrenergic receptor agonist, clenbuterol on hepatic autophagy. Surprisingly, we found that clenbuterol stimulated autophagy and autophagic flux in hepatoma cells, primary hepatocytes and in vivo. Similar effects also were observed with epinephrine treatment. Interestingly, propranolol caused a late block in autophagy in the absence and presence of clenbuterol, both in cell culture and in vivo. Thus, our results demonstrate that the β2-adrenergic receptor is a key regulator of hepatic autophagy, and that the β-blocker propranolol can independently induce a late block in autophagy.

  14. Yes-associated protein/TEA domain family member and hepatocyte nuclear factor 4-alpha (HNF4α) repress reciprocally to regulate hepatocarcinogenesis in rats and mice.

    PubMed

    Cai, Wang-Yu; Lin, Ling-Yun; Hao, Han; Zhang, Sai-Man; Ma, Fei; Hong, Xin-Xin; Zhang, Hui; Liu, Qing-Feng; Ye, Guo-Dong; Sun, Guang-Bin; Liu, Yun-Jia; Li, Sheng-Nan; Xie, Yuan-Yuan; Cai, Jian-Chun; Li, Bo-An

    2017-04-01

    Great progress has been achieved in the study of Hippo signaling in regulating tumorigenesis; however, the downstream molecular events that mediate this process have not been completely defined. Moreover, regulation of Hippo signaling during tumorigenesis in hepatocellular carcinoma (HCC) remains largely unknown. In the present study, we systematically investigated the relationship between Yes-associated protein/TEA domain family member (YAP-TEAD) and hepatocyte nuclear factor 4-alpha (HNF4α) in the hepatocarcinogenesis of HCC cells. Our results indicated that HNF4α expression was negatively regulated by YAP1 in HCC cells by a ubiquitin proteasome pathway. By contrast, HNF4α was found to directly associate with TEAD4 to compete with YAP1 for binding to TEAD4, thus inhibiting the transcriptional activity of YAP-TEAD and expression of their target genes. Moreover, overexpression of HNF4α was found to significantly compromise YAP-TEAD-induced HCC cell proliferation and stem cell expansion. Finally, we documented the regulatory mechanism between YAP-TEAD and HNF4α in rat and mouse tumor models, which confirmed our in vitro results.

  15. Retinoic acid regulates several genes in bile acid and lipid metabolism via upregulation of small heterodimer partner in hepatocytes.

    PubMed

    Mamoon, Abulkhair; Subauste, Angela; Subauste, Maria C; Subauste, Jose

    2014-10-25

    Retinoic acid (RA) affects multiple aspects of development, embryogenesis and cell differentiation processes. The liver is a major organ that stores RA suggesting that retinoids play an important role in the function of hepatocytes. In our previous studies, we have demonstrated the involvement of small heterodimer partner (SHP) in RA-induced signaling in a non-transformed hepatic cell line AML 12. In the present study, we have identified several critical genes in lipid homeostasis (Apoa1, Apoa2 and ApoF) that are repressed by RA-treatment in a SHP dependent manner, in vitro and also in vivo with the use of the SHP null mice. In a similar manner, RA also represses several critical genes involved in bile acid metabolism (Cyp7a1, Cyp8b1, Mdr2, Bsep, Baat and Ntcp) via upregulation of SHP. Collectively our data suggest that SHP plays a major role in RA-induced potential changes in pathophysiology of metabolic disorders in the liver.

  16. Glucagon and cAMP inhibit cholesterol 7alpha-hydroxylase (CYP7A1) gene expression in human hepatocytes: discordant regulation of bile acid synthesis and gluconeogenesis.

    PubMed

    Song, Kwang-Hoon; Chiang, John Y L

    2006-01-01

    The gene encoding cholesterol 7alpha-hydroxylase (CYP7A1) is tightly regulated to control bile acid synthesis and maintain lipid homeostasis. Recent studies in mice suggest that bile acid synthesis is regulated by the fasted-to-fed cycle, and fasting induces CYP7A1 gene expression in parallel to the induction of peroxisome proliferators-activated receptor gamma co-activator 1alpha (PGC-1alpha) and phosphoenolpyruvate carboxykinase (PEPCK). How glucagon regulates CYP7A1 gene expression in the human liver is not clear. Here we show that glucagon and cyclic adenosine monophosphate (cAMP) strongly repressed CYP7A1 mRNA expression in human primary hepatocytes. Reporter assays confirmed that cAMP and protein kinase A (PKA) inhibited human CYP7A1 gene transcription, in contrast to their stimulation of the PEPCK gene. Mutagenesis analysis identified a PKA-responsive region located within the previously identified HNF4alpha binding site in the human CYP7A1 promoter. Glucagon and cAMP increased HNF4alpha phosphorylation and reduced the amount of HNF4alpha present in CYP7A1 chromatin. Our findings suggest that glucagon inhibited CYP7A1 gene expression via PKA phosphorylation of HNF4alpha, which lost its ability to bind the CYP7A1 gene and resulted in inhibition of human CYP7A1 gene transcription. In conclusion, this study unveils a species difference in nutrient regulation of the human and mouse CYP7A1 gene and suggests a discordant regulation of bile acid synthesis and gluconeogenesis by glucagon in human livers during fasting.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. Frequency of mutation to rifampin resistance in Streptococcus pneumoniae clinical strains: hexA and hexB polymorphisms do not account for hypermutation.

    PubMed

    Morosini, María-Isabel; Baquero, María-Rosario; Sánchez-Romero, J M; Negri, María-Cristina; Galán, Juan-Carlos; del Campo, Rosa; Pérez-Díaz, J C; Baquero, Fernando

    2003-04-01

    The frequency of mutation to rifampin resistance of 200 clinical Streptococcus pneumoniae isolates was examined. Two peaks were observed in the distribution, with mode frequencies of 2.5 x 10(-7) (20% of isolates) and 2.5 x 10(-8). The hexA and hexB gene entire sequences were analyzed in 13 isolates. Sequences from both hypermutable and "normomutable" strains were conserved relative to that of the R6 S. pneumoniae control strain. The phenotypic Hex system proficiency, in terms of transforming efficiency, was also maintained irrespective of the variations in mutation frequency values.

  5. Lipid Regulation Effects of Raw and Processed Notoginseng Radix Et Rhizome on Steatotic Hepatocyte L02 Cell.

    PubMed

    Chen, Zhu; Li, Chunmei; Yang, Caixia; Zhao, Ronghua; Mao, Xiaojian; Yu, Jie

    2016-01-01

    Introduction. Raw and processed Notoginseng Radix Et Rhizome (NRR) have been widely used in treatment of metabolic syndromes and related disease, including nonalcoholic fatty liver disease (NAFLD). This study was designed to investigate lipid regulation effects of raw and processed NRR in steatotic L02 cell. Materials and Methods. Steatotic L02 cells were obtained after being cultured with 5% fat emulsion-10% FBS-RPMI 1640 medium for 48 h. Contents of total cholesterol (TC), triglyceride (TG), free fatty acid (FFA), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in steatotic L02 cells were evaluated after treatment. Furthermore, the lipid metabolism regulation mechanism of Panax notoginseng saponins (PNS) and its monomers were evaluated by detecting the expressions of hydroxymethyl glutaric acyl coenzyme A reductase (HMG-CoAR), sterol regulating element binding protein-2 (SREBP-2), and cholesterol 7α-hydroxylase (CYP7α). Results. TG and TC contents were doubled in model group compared to those in normal L02 cells group. Raw NRR and NRR heated with sand (NRR-B) showed much remarkable lipid-lowering effects in steatotic L02 cells. PNS, notoginsenoside R1, ginsenoside Rg1, and ginsenoside Rb1 displayed the best TG and TC regulation activity, which could significantly reduce contents of SREBP-2 and HMG-CoAR and increase the content of CYP7α. Conclusions. Our results may support the fact that both raw NRR and NRR-B might have more satisfactory effects in the treatment of NAFLD.

  6. Lipid Regulation Effects of Raw and Processed Notoginseng Radix Et Rhizome on Steatotic Hepatocyte L02 Cell

    PubMed Central

    Li, Chunmei; Yang, Caixia; Zhao, Ronghua

    2016-01-01

    Introduction. Raw and processed Notoginseng Radix Et Rhizome (NRR) have been widely used in treatment of metabolic syndromes and related disease, including nonalcoholic fatty liver disease (NAFLD). This study was designed to investigate lipid regulation effects of raw and processed NRR in steatotic L02 cell. Materials and Methods. Steatotic L02 cells were obtained after being cultured with 5% fat emulsion-10% FBS-RPMI 1640 medium for 48 h. Contents of total cholesterol (TC), triglyceride (TG), free fatty acid (FFA), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in steatotic L02 cells were evaluated after treatment. Furthermore, the lipid metabolism regulation mechanism of Panax notoginseng saponins (PNS) and its monomers were evaluated by detecting the expressions of hydroxymethyl glutaric acyl coenzyme A reductase (HMG-CoAR), sterol regulating element binding protein-2 (SREBP-2), and cholesterol 7α-hydroxylase (CYP7α). Results. TG and TC contents were doubled in model group compared to those in normal L02 cells group. Raw NRR and NRR heated with sand (NRR-B) showed much remarkable lipid-lowering effects in steatotic L02 cells. PNS, notoginsenoside R1, ginsenoside Rg1, and ginsenoside Rb1 displayed the best TG and TC regulation activity, which could significantly reduce contents of SREBP-2 and HMG-CoAR and increase the content of CYP7α. Conclusions. Our results may support the fact that both raw NRR and NRR-B might have more satisfactory effects in the treatment of NAFLD. PMID:27642594

  7. Folate Protects Hepatocytes of Hyperhomocysteinemia Mice from Apoptosis via Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-activated Endoplasmic Reticulum Stress.

    PubMed

    Yang, Anning; Sun, Yue; Mao, Caiyan; Yang, Songhao; Huang, Min; Deng, Mei; Ding, Ning; Yang, Xiaoling; Zhang, Minghao; Jin, Shaoju; Jiang, Yideng; Huang, Ying

    2017-02-23

    Folate deficiency is a known risk factor for liver injury; however, the underlying mechanism remains unclear. In this study, we employed a high homocysteine-induced liver injury model of Apolipoprotein E-deficient (ApoE(-/-) ) mice fed high-methionine diet and found that high homocysteine induced endoplasmic reticulum (ER) stress and liver cell apoptosis by downregulation of cystic fibrosis transmembrane conductance regulator (CFTR) expression; observations that were attenuated with supplementation of dietary folate. The regulation on CFTR expression was mediated by CFTR promoter methylation and trimethylation of lysine 27 on histone H3 (H3K27me3). Mechanistically, folate inhibited homocysteine-induced CFTR promoter methylation and H3K27me3, which resulted in upregulation of CFTR expression, and reduced ER stress and liver cell apoptosis. Further study showed that folate inhibited the expression of DNA methyltransferase 1 and enhancer of zeste homolog 2, downregulated the cellular concentrations of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) and upregulated the SAM/SAH ratio, leading to the inhibition of Hcy-induced DNA hypermethylation and H3K27me3 in CFTR promoter. In conclusion, our results provide insight into the protective role of folate in homocysteine-induced ER stress and liver cell apoptosis through the regulation of CFTR expression. This article is protected by copyright. All rights reserved.

  8. Hepatocytes: critical for glucose homeostasis.

    PubMed

    Klover, Peter J; Mooney, Robert A

    2004-05-01

    Maintaining blood glucose levels within a narrow range is a critical physiological function requiring multiple metabolic pathways and involving several cell types, including a prominent role for hepatocytes. Under hormonal control, hepatocytes can respond to either feeding or fasting conditions by storing or producing glucose as necessary. In the fasting state, the effects of glucagon avoid hypoglycemia by stimulating glucogenesis and glycogenolysis and initiating hepatic glucose release. Postprandially, insulin prevents hyperglycemia, in part, by suppressing hepatic gluconeogenesis and glycogenolysis and facilitating hepatic glycogen synthesis. Both transcriptional regulation of rate limiting enzymes and modulation of enzyme activity through phosphorylation and allosteric regulation are involved. Type 2 diabetes mellitus is the most common serious metabolic condition in the world, and results from a subnormal response of tissues to insulin (insulin resistance) and a failure of the insulin-secreting beta cells to compensate. In type 2 diabetes, glucose is overproduced by the hepatocyte and is ineffectively metabolized by other organs. Impairments in the insulin signal transduction pathway appear to be critical lesions contributing to insulin resistance and type 2 diabetes.

  9. 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

  10. 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

  11. Mismatch repair genes of Streptococcus pneumoniae: HexA confers a mutator phenotype in Escherichia coli by negative complementation.

    PubMed

    Prudhomme, M; Méjean, V; Martin, B; Claverys, J P

    1991-11-01

    DNA repair systems able to correct base pair mismatches within newly replicated DNA or within heteroduplex molecules produced during recombination are widespread among living organisms. Evidence that such generalized mismatch repair systems evolved from a common ancestor is particularly strong for two of them, the Hex system of the gram-positive Streptococcus pneumoniae and the Mut system of the gram-negative Escherichia coli and Salmonella typhimurium. The homology existing between HexA and MutS and between HexB and MutL prompted us to investigate the effect of expressing hex genes in E. coli. Complementation of mutS or mutL mutations, which confer a mutator phenotype, was assayed by introducing on a multicopy plasmid the hexA and hexB genes, under the control of an inducible promoter, either individually or together in E. coli strains. No decrease in mutation rate was conferred by either hexA or hexB gene expression. However, a negative complementation effect was observed in wild-type E. coli cells: expression of hexA resulted in a typical Mut- mutator phenotype. hexB gene expression did not increase the mutation rate either individually or in conjunction with hexA. Since expression of hexA did not affect the mutation rate in mutS mutant cells and the hexA-induced mutator effect was recA independent, it is concluded that this effect results from inhibition of the Mut system. We suggest that HexA, like its homolog MutS, binds to mismatches resulting from replication errors, but in doing so it protects them from repair by the Mut system. In agreement with this hypothesis, an increase in mutS gene copy number abolished the hexA-induced mutator phenotype. HexA protein could prevent repair either by being unable to interact with Mut proteins or by producing nonfunctional repair complexes.

  12. Hex-square moire patterns in imagers using microchannel plates

    NASA Technical Reports Server (NTRS)

    Lawrence, George M.

    1989-01-01

    In electronic imaging detectors using microchannel plates, the mismatch between the pixels on a square mesh and the microchannels on a hexagonal mesh produces moire image defects. Theoretical statistical estimates of the sizes of the microposition offsets and the flat field intensity errors are calculated, showing the trade-off between resolution and position accuracy. A distinction is made between moments of spot images and moments of the single-pixel-response functions. As the resolution between the hex and square meshes is improved, the detector resolution is improved, but at the expense of an about 10 percent moire pattern. These moire patterns will not be properly corrected by dividing by the flat field image.

  13. Mechanism of Hepatocyte Apoptosis

    PubMed Central

    Cao, Lei; Quan, Xi-Bing; Zeng, Wen-Jiao; Yang, Xiao-Ou; Wang, Ming-Jie

    2016-01-01

    Hepatocyte apoptosis plays important roles in both the removal of external microorganisms and the occurrence and development of liver diseases. Different conditions, such as virus infection, fatty liver disease, hepatic ischemia reperfusion, and drug-induced liver injury, are accompanied by hepatocyte apoptosis. This review summarizes recent research on the mechanism of hepatocyte apoptosis involving the classical extrinsic and intrinsic apoptotic pathways, endoplasmic reticulum stress, and oxidative stress-induced apoptosis. We emphasized the major causes of apoptosis according to the characteristics of different liver diseases. Several concerns regarding future research and clinical application are also raised. PMID:28058033

  14. Evaluation of UGT protein interactions in human hepatocytes: Effect of siRNA down regulation of UGT1A9 and UGT2B7 on propofol glucuronidation in human hepatocytes☆

    PubMed Central

    Konopnicki, Camille M.; Dickmann, Leslie J.; Tracy, Jeffrey M.; Tukey, Robert H.; Wienkers, Larry C.; Foti, Robert S.

    2014-01-01

    Previous experiments performed in recombinant systems have suggested that protein–protein interactions occur between the UGTs and may play a significant role in modulating enzyme activity. However, evidence of UGT protein–protein interactions either in vivo or in more physiologically relevant in vitro systems has yet to be demonstrated. In this study, we examined oligomerization and its ability to affect glucuronidation in plated human hepatocytes. siRNA down regulation experiments and activity studies were used to examine changes in metabolite formation of one UGT isoform due to down regulation of a second UGT isoform. Selective siRNA directed towards UGT1A9 or UGT2B7 resulted in significant and selective decreases in their respective mRNA levels. As expected, the metabolism of the UGT1A9 substrate propofol decreased with UGT1A9 down regulation. Interestingly, UGT1A9 activity, but not UGT1A9 mRNA expression, was also diminished when UGT2B7 expression was selectively inhibited, implying potential interactions between the two isoforms. Minor changes to UGT1A4, UGT2B4 and UGT2B7 activity were also observed when UGT1A9 expression was selectively down regulated. To our knowledge, this represents the first piece of evidence that UGT protein–protein interactions occur in human hepatocytes and suggests that expression levels of UGT2B7 may directly impact the glucuronidation activity of selective UGT1A9 substrates. PMID:23562620

  15. Increased Expression of Hepatocyte Nuclear Factor 6 Stimulates Hepatocyte Proliferation during Mouse Liver Regeneration

    PubMed Central

    Tan, Yongjun; Yoshida, Yuichi; Hughes, Douglas E.; Costa, Robert H.

    2005-01-01

    Background & Aims The Hepatocyte Nuclear Factor 6 (HNF6 or ONECUT-1) protein is a cell-type specific transcription factor that regulates expression of hepatocyte-specific genes. Using hepatocytes for Chromatin Immunoprecipitation (ChIP) assays, the HNF6 protein was shown to associate with cell cycle regulatory promoters. Here, we examined whether increased levels of HNF6 stimulate hepatocyte proliferation during mouse liver regeneration. Methods Tail vein injection of adenovirus expressing the HNF6 cDNA (AdHNF6) was used to increase hepatic HNF6 levels during mouse liver regeneration induced by partial hepatectomy, and DNA replication was determined by Bromodeoxyuridine incorporation. Cotransfection and ChIP assays were used to determine transcriptional target promoters. Results Elevated expression of HNF6 during mouse liver regeneration causes a significant increase in the number of hepatocytes entering DNA replication (S-phase) and mouse hepatoma Hepa1-6 cells diminished for HNF6 levels by siRNA transfection exhibit a 50% reduction in S-phase following serum stimulation. This stimulation in hepatocyte S-phase progression was associated with increased expression of the hepatocyte mitogen Tumor Growth Factor α (TGFα) and the cell cycle regulators Cyclin D1 and Forkhead Box m1 (Foxm1) transcription factor. Cotransfection and ChIP assays show that TGFα, Cyclin D1, and HNF6 promoter regions are direct transcriptional targets of the HNF6 protein. Co-immunoprecipitation assays with regenerating mouse liver extracts reveal association between HNF6 and Foxm1 proteins and cotransfection assays show that HNF6 stimulates Foxm1 transcriptional activity. Conclusion These mouse liver regeneration studies show that increased HNF6 levels stimulate hepatocyte proliferation through transcriptional induction of cell cycle regulatory genes. PMID:16618419

  16. 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

  17. Regulation of cytochrome P450 expression by inhibitors of hydroxymethylglutaryl-coenzyme A reductase in primary cultured rat hepatocytes and in rat liver.

    PubMed

    Kocarek, T A; Reddy, A B

    1996-11-01

    It was previously demonstrated that treatment of primary cultured rat hepatocytes with lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, induced the mRNAs for several cytochromes P450 (P450s), including CYP2B1/2, CYP3A1/2, and CYP4A. In this study, we have compared the effects of lovastatin with those of three additional HMG-CoA reductase inhibitors (simvastatin, pravastatin, and the structurally dissimilar drug fluvastatin) on P450 expression in primary cultured rat hepatocytes, and we have also characterized the effects of in vivo treatment with fluvastatin on P450 expression in rat liver. Treatment of cultured hepatocytes with lovastatin, simvastatin, or fluvastatin increased CYP2B1/2, CYP3A1/2, and CYP4A mRNA and immunoreactive protein levels over the dose range (3 x 10(-6) to 3 x 10(-5) M) required to increase the amount of HMG-CoA reductase mRNA. The increases in CYP2B1/2 levels produced by 3 x 10(-5) M fluvastatin treatment were larger than those produced by lovastatin or simvastatin treatment or by treatment with 10(-4) M phenobarbital. In contrast, treatment of cultured hepatocytes with 3 x 10(-5) M lovastatin, simvastatin, or fluvastatin increased CYP3A1/2 and CYP4A mRNA and immunoreactive protein to lower levels than those produced by treatment with 10(-5) M dexamethasone or 10(-4) M ciprofibrate. Treatment of cultured hepatocytes with pravastatin had little or no effect on the amount of any of the P450s examined, although this drug induced HMG-CoA reductase mRNA as effectively as did fluvastatin. Incubation of hepatocytes with 10(-4) M fluvastatin increased CYP1A1 mRNA to 67% of the level induced by treatment with 10(-5) M beta-naphthoflavone. Doses of 50 or 100 mg/ kg/day fluvastatin administered for 3 days to rats increased the hepatic levels of CYP2B1/2 and CYP4A mRNA and immunoreactive protein, although to much lower levels than those produced by treatment with phenobarbital or ciprofibrate, respectively. Treatment of

  18. HEX: a novel homeobox gene expressed during haematopoiesis and conserved between mouse and human.

    PubMed Central

    Bedford, F K; Ashworth, A; Enver, T; Wiedemann, L M

    1993-01-01

    We describe the cloning of a novel homeodomain-containing gene, which is highly conserved between mouse and human. The human cDNA was initially isolated from human haematopoietic tissue and denoted HEX (haematopoietically expressed homeobox). Sequence analysis of the coding sequences from mouse and the partial cDNA from human shows that the homeodomain is most closely related to those of the HIx and HOX11 proteins. The HEX gene is present as a single copy in the human genome. Analysis of murine genomic DNA shows, in addition to an intron-containing gene homologous to HEX, the presence of a processed copy of the gene which has arisen within the last few million years. Analysis of human and murine haematopoietic cells and cell lines, revealed expression of the HEX gene in multipotential progenitors, as well as cells of the B-lymphocyte and myeloid lineages. However HEX was not expressed in T-lymphocytes or erythroid cells. This pattern of HEX gene expression suggests that it may play a role in haematopoietic differentiation. Images PMID:8096636

  19. Hepatocyte membrane water permeability measured by silicone layer filtering centrifugation.

    PubMed

    Gradilone, Sergio A; Ochoa, J Elena; García, Fabiana; Larocca, M Cecilia; Pellegrino, José M; Marinelli, Raúl A

    2002-03-01

    We previously found that hepatocytes are able to control their osmotic membrane water permeability (P(f)) by regulating the number of surface aquaporin water channels. Hepatocyte P(f) has been assessed by phase-contrast microscopy and cell image analysis, an established but relatively laborious procedure. We report here an alternative method to assess hepatocyte P(f) based on a single silicone layer filtering centrifugation system. Isolated rat hepatocytes were incubated in hypotonic or isotonic buffers containing (3)H(2)O as a tracer and, then, were filtered by rapid centrifugation through a silicone layer down to a lysis layer. Osmotically driven radioactivity (i.e., (3)H(2)O) within hepatocytes was calculated as the difference between the dpm in lysis media measured under hypotonic and isotonic conditions. The P(f) calculated from the initial slope of the radioactivity-versus-time curve was 18 microm/s at 4 degrees C. Hepatocytes treated with dibutyryl cyclic AMP, to increase P(f) through the plasma membrane insertion of aquaporins, showed an increased P(f) value of 37 microm/s. The aquaporin blocker dimethyl sulfoxide selectively prevented the agonist-induced hepatocyte P(f). These data are in good agreement with the corresponding values determined by quantitative phase-contrast microscopy; thus, the method developed allows the rapid and reliable measurement of hepatocyte P(f).

  20. The comparative effects of diethyldithiocarbamate-copper complex with established proteasome inhibitors on expression levels of CYP1A2/3A4 and their master regulators, aryl hydrocarbon and pregnane X receptor in primary cultures of human hepatocytes.

    PubMed

    Vrzal, Radim; Dvorak, Zdenek

    2016-12-01

    In the recent years, a therapeutic potential of disulfiram (Antabuse) complex with copper, as an anticancer drug, was recognized towards several cancer cell lines. The proteasome was suggested as one of the cellular targets for this compound. As the therapeutic use of diethyldithiocarbamate-copper complex (CuET) is expected to increase, it is of great interest to know whether this compound may be the source of drug-drug interactions via the induction of biotransformation enzymes, especially cytochromes P450 (CYPs). To this purpose, we examined the effect of CuET and compared it with typical inducers (rifampicin and dioxin) of CYPs and with well-established proteasome inhibitors (MG132 and bortezomib). Diethyldithiocarbamate-copper complex revealed inconsistent and rather modulatory effect on the expression of CYP1A2 and CYP3A4 in several cultures of human hepatocytes. Moreover, it was able to cause neither ubiquitin accumulation nor significant and dose-dependent inhibition of proteasome activity. It had no effect on essential transcription factors involved in regulation of selected CYPs, aryl hydrocarbon (AhR) nor pregnane X receptor (PXR). However, the AhR protein was increased in majority of examined hepatocyte cultures. The main finding of this study is that: (i) disulfiram-copper complex is not the cause of drug-drug interactions via CYP1A2/3A4 induction; (ii) proteasome inhibitors may have different impact on studied parameters in given in vitro system.

  1. Nucleotide sequence of the hexA gene for DNA mismatch repair in Streptococcus pneumoniae and homology of hexA to mutS of Escherichia coli and Salmonella typhimurium

    SciTech Connect

    Priebe, S.D.; Hadi, S.M.; Greenberg, B.; Lacks, S.A.

    1988-01-01

    The Hex system of heteroduplex DNA base mismatch repair operates in Streptococcus pneumoniae after transformation and replication to correct donor and nascent DNA strands, respectively. A functionally similar system, called Mut, operates in Escherichia coli and Salmonella typhimurium. The nucleotide sequence of a 3.8-kilobase segment from the S. pneumoniae chromosome that includes the 2.7-kilobase hexA gene was determined. Chromosomal DNA used as donor to measure Hex phenotype was irradiated with UV light. An open reading frame that could encode a 17-kilodalton polypeptide (OrfC) was located just upstream of the gene encoding a polypeptide of 95 kilodaltons corresponding to HexA. Shine-Dalgarno sequences and putative promoters were identified upstream of each protein start site. Insertion mutations showed that only HexA functioned in mismatch repair and that the promoter for hexA transcription was located within the OrfC-coding region. The HexA polypeptide contains a consensus sequence for ATP- or GTP-binding sites in proteins. Comparison of the entire HexA protein sequence to that of MutS of S. typhimurium, showed the proteins to be homologous, inasmuch as 36% of their amino acid residues were identical. This homology indicates that the Hex and Mut systems of mismatch repair evolved from an ancestor common to the gram-positive streptococci and the gram-negative enterobacteria. It is the first direct evidence linking the two systems.

  2. Cloning of the hexA mismatch-repair gene of Streptococcus pneumoniae and identification of the product.

    PubMed

    Martin, B; Prats, H; Claverys, J P

    1985-01-01

    The hexA mismatch repair gene of Streptococcus pneumoniae has been cloned into multicopy plasmid vectors. The cloned hexA gene is expressed as judged from its ability to complement various chromosomal hexA- alleles. Its direction of transcription was defined and the functional limits were localized by original methods relying on homology-dependent integration of nonautonomous chimeric plasmids carrying chromosomal inserts into the chromosome. Comparison of the proteins encoded by recombinant plasmids and by restriction fragments allowed us to identify an Mr 94 000 protein as the probable product of the hexA gene.

  3. Design of a Vector-Vorticity Dynamical Core on a Hexagonal Grid (Hex-VVDC)

    NASA Astrophysics Data System (ADS)

    Konor, C. S.; Heikes, R. P.; Jung, J.; Randall, D.; Arakawa, A.

    2009-05-01

    We are developing a Global Cloud-Resolving Model (GCRM) based on the vector-vorticity dynamical core (VVDC) that predicts three-dimensional vorticity. The VVDC in an anelastic framework has been originally designed by Jung and Arakawa and used in a Cartesian grid. As the first step in developing the GCRM, we have constructed an intermediate model with the VVDC using a hexagonal grid on a planar quasi-rectangular horizontal domain, which we call the hexagonal VVDC (Hex-VVDC). The purpose of developing the Hex-VVDC is to test and verify the discretization of equations of the VVDC on a hexagonal grid before applying them to the geodesic hexagon/pentagon grid that will be used for the GCRM. It is found that having twice as many as cell walls and three times as many as cell corners than the cell centers in a hexagonal grid produces a computational mode in horizontal and vertical components of vorticity and horizontal velocity fields, which the dynamics cannot control. We have designed a special scheme to treat this computational mode. With this treatment, the Hex-VVDC seems to work well. Currently, we are performing further tests with the Hex-VVDC and also continuing the application of the dynamical core to the geodesic hexagon/pentagon grid. In this presentation, we will discuss the computational design of the Hex-VVDC and show simulated results primarily focusing on vertical transports of vorticity and momentum by convection.

  4. 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

  5. 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.

  6. 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.

  7. Characterization and selection of CZT detector modules for HEX experiment onboard Chandrayaan-1

    NASA Astrophysics Data System (ADS)

    Vadawale, S. V.; Purohit, S.; Shanmugam, M.; Acharya, Y. B.; Goswami, J. N.; Sudhakar, M.; Sreekumar, P.

    2009-01-01

    We present the results of characterization of a large sample of Cadmium Zinc Telluride (CZT) detector modules planned to be used for the HEX (High Energy X-ray spectrometer) experiment onboard India's first mission to the Moon, Chandrayaan-1. We procured forty modules from Orbotech Medical Solutions Ltd. and carried out a detailed characterization of each module at various temperatures and selected final nine detector modules for the flight model of HEX. Here we present the results of the characterization of all modules and the selection procedure for the HEX flight detector modules. These modules show 5-6% energy resolution (at 122 keV, for best 90% of pixels) at room temperature which is improved to ˜4% when these modules are cooled to sub-0 °C temperature. The gain and energy resolution were stable during the long duration tests.

  8. WITS-HEX: A renovated code for analysis of PIXE X-ray spectra

    NASA Astrophysics Data System (ADS)

    Lipworth, A. D.; Annegarn, H. J.; Bauman, S.; Molokomme, T.; Walker, A. J.

    1990-04-01

    The code REX [H.C. Kaufmann and K.R. Akselsson, Adv. X-ray Anal. 18 (1975) 353] and its successor, HEX were developed at Florida State University and University of Lund in the early seventies for quantitative reduction of PIXE spectra. HEX modelled many of the physical processes, including target self-absorption. Lack of adequate documentation and several inherent software related problems hampered widespread adoption and further development. HEX was designed using a hierarchical functional design approach, coded in FORTRAN IV. Its element library and request list are tightly constrained. ANSI standard FORTRAN provides few structured constructs, nor does it support dynamic data structures. HEX has a batch mode of interaction which affords minimal (interim) user interactions. To overcome these limitations we have modernised the program to run on IBM PC compatible computers by using object-oriented design techniques, documented in Program Description Language (pseudo-code) and implemented in a modern programming language, Pascal. Turbo Pascal 5.5 provides an integrated program development environment, a high resolution graphics library, an operating system interface and many software development tools to improve programmer productivity. A menu-based highly interactive screen management library is used for the user interface. A demonstration version is available for user evaluation. Further rigorous testing and the development of user acquired spectra interfacing code will be developed before the first release. The renovated code, WITS-HEX, has extracted the intellectual value embodied in HEX. It will be inherently more stable, reliable, versatile, maintainable and easy to use to facilitate future process modifications.

  9. Draft Genome Sequence of Pseudomonas aeruginosa Strain Hex1T Isolated from Soils Contaminated with Used Lubricating Oil in Argentina

    PubMed Central

    Luján, Adela M.; Feliziani, Sofía

    2017-01-01

    ABSTRACT Pseudomonas aeruginosa Hex1T was isolated from soils contaminated with used lubricating oil from a garage in Córdoba, Argentina. This strain is capable of utilizing this pollutant as the sole carbon and energy source. Here, we present the 6.9-Mb draft genome sequence of Hex1T, which contains many heavy metal-resistance genes. PMID:28082504

  10. Regulation of constitutive androstane receptor and its target genes by fasting, cAMP, hepatocyte nuclear factor alpha, and the coactivator peroxisome proliferator-activated receptor gamma coactivator-1alpha.

    PubMed

    Ding, Xunshan; Lichti, Kristin; Kim, Insook; Gonzalez, Frank J; Staudinger, Jeff L

    2006-09-08

    Animal studies reveal that fasting and caloric restriction produce increased activity of specific metabolic pathways involved in resistance to weight loss in liver. Evidence suggests that this phenomenon may in part occur through the action of the constitutive androstane receptor (CAR, NR1I3). Currently, the precise molecular mechanisms that activate CAR during fasting are unknown. We show that fasting coordinately induces expression of genes encoding peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha), CAR, cytochrome P-450 2b10 (Cyp2b10), UDP-glucuronosyltransferase 1a1 (Ugt1a1), sulfotransferase 2a1 (Sult2a1), and organic anion-transporting polypeptide 2 (Oatp2) in liver in mice. Treatments that elevate intracellular cAMP levels also produce increased expression of these genes in cultured hepatocytes. Our data show that PGC-1alpha interaction with hepatocyte nuclear factor 4alpha (HNF4alpha, NR2A1) directly regulates CAR gene expression through a novel and evolutionarily conserved HNF4-response element (HNF4-RE) located in its proximal promoter. Expression of PGC-1alpha in cells increases CAR expression and ligand-independent CAR activity. Genetic studies reveal that hepatic expression of HNF4alpha is required to produce fasting-inducible CAR expression and activity. Taken together, our data show that fasting produces increased expression of genes encoding key metabolic enzymes and an uptake transporter protein through a network of interactions involving cAMP, PGC-1alpha, HNF4alpha, CAR, and CAR target genes in liver. Given the recent finding that mice lacking CAR exhibit a profound decrease in resistance to weight loss during extended periods of caloric restriction, our findings have important implications in the development of drugs for the treatment of obesity and related diseases.

  11. 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.

  12. 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.

  13. A Ca(2+) chelator ameliorates chromium (VI)-induced hepatocyte L-02 injury via down-regulation of voltage-Dependent anion channel 1 (VDAC1) expression.

    PubMed

    Yi, Xing; Xiao, Fang; Zhong, Xiali; Duan, Yujie; Liu, Kaihua; Zhong, Caigao

    2017-01-01

    Hexavalent chromium could result in cell malfunctions. Intracellular Ca(2+) ([Ca(2+)]i) content and VDAC1 expression are both important features related to cell survial. This study aimed to explore the mechanism of cell injury induced by Cr(VI) and tentatively offer clues to repairing this cell damage using [Ca(2+)]i and VDAC1. L-02 hepatocytes were treated with Cr(VI)/BAPTA, and the levels of [Ca(2+)]i and cell injury associated with Cr(VI) were determined in addition to the effect of BAPTA. The expression of VDAC1 in Cr(VI)-induced cells was evaluated. The results showed a dose-dependent elevation of the level of VDAC1 and the mRNA level of the VDAC1 biogenesis-related gene Sam50. BAPTA could ameliorate less severe damage induced by 4μM Cr(VI) via reducing VDAC1 and Sam50. Additionally, cell injury caused by less than 4μM Cr(VI) could be ameliorated by VDAC1 knockdown. Taken together, the findings of this study suggest that inhibition of intracellular Ca(2±) overload could protect cells from damage and that VDAC1 plays a considerable role in Cr(VI)-induced liver injury.

  14. Neuroprotective Efficacy from a Lipophilic Redox-Modulating Mn(III) N-Hexylpyridylporphyrin, MnTnHex-2-PyP: Rodent Models of Ischemic Stroke and Subarachnoid Hemorrhage

    PubMed Central

    Sheng, Huaxin; Spasojevic, Ivan; Tse, Hubert M.; Jung, Jin Yong; Hong, Jun; Zhang, Zhiquan; Piganelli, Jon D.; Batinic-Haberle, Ines

    2011-01-01

    Intracerebroventricular treatment with redox-regulating Mn(III) N-hexylpyridylporphyrin (MnPorphyrin) is remarkably efficacious in experimental central nervous system (CNS) injury. Clinical development has been arrested because of poor blood-brain barrier penetration. Mn(III) meso-tetrakis (N-hexylpyridinium-2-yl) porphyrin (MnTnHex-2-PyP) was synthesized to include four six-carbon (hexyl) side chains on the core MnPorphyrin structure. This has been shown to increase in vitro lipophilicity 13,500-fold relative to the hydrophilic ethyl analog Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP). In normal mice, we found brain MnTnHex-2-PyP accumulation to be ∼9-fold greater than MnTE-2-PyP 24 h after a single intraperitoneal dose. We then evaluated MnTnHex-2-PyP efficacy in outcome-oriented models of focal cerebral ischemia and subarachnoid hemorrhage. For focal ischemia, rats underwent 90-min middle cerebral artery occlusion. Parenteral MnTnHex-2-PyP treatment began 5 min or 6 h after reperfusion onset and continued for 7 days. Neurologic function was improved with both early (P = 0.002) and delayed (P = 0.002) treatment onset. Total infarct size was decreased with both early (P = 0.03) and delayed (P = 0.01) treatment. MnTnHex-2-PyP attenuated nuclear factor κB nuclear DNA binding activity and suppressed tumor necrosis factor-α and interleukin-6 expression. For subarachnoid hemorrhage, mice underwent perforation of the anterior cerebral artery and were treated with intraperitoneal MnTnHex-2-PyP or vehicle for 3 days. Neurologic function was improved (P = 0.02), and vasoconstriction of the anterior cerebral (P = 0.0005), middle cerebral (P = 0.003), and internal carotid (P = 0.015) arteries was decreased by MnTnHex-2-PyP. Side-chain elongation preserved MnPorphyrin redox activity, but improved CNS bioavailability sufficient to cause improved outcome from acute CNS injury, despite delay in parenteral treatment onset of up to 6 h. This advance now

  15. 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...

  16. 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

    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.

  17. Biomechanics and functionality of hepatocytes in liver cirrhosis.

    PubMed

    Sun, Shan; Song, Zhenyuan; Cotler, Scott J; Cho, Michael

    2014-06-27

    Cirrhosis is a life-threatening condition that is generally attributed to overproduction of collagen fibers in the extracellular matrix that mechanically stiffens the liver. Chronic liver injury due to causes including viral hepatitis, inherited and metabolic liver diseases and external factors such as alcohol abuse can result in the development of cirrhosis. Progression of cirrhosis leads to hepatocellular dysfunction. While extensive studies to understand the complexity underlying liver fibrosis have led to potential application of anti-fibrotic drugs, no such FDA-approved drugs are currently available. Additional studies of hepatic fibrogenesis and cirrhosis primarily have focused on the extracellular matrix, while hepatocyte biomechanics has received limited attention. The role of hepatocyte biomechanics in liver cirrhosis remains elusive, and how the cell stiffness is correlated with biological functions of hepatocytes is also unknown. In this study, we demonstrate that the biomechanical properties of hepatocytes are correlated with their functions (e.g., glucose metabolism), and that hepatic dysfunction can be restored through modulation of the cellular biomechanics. Furthermore, our results indicate the hepatocyte functionality appears to be regulated through a crosstalk between the Rho and Akt signaling. These novel findings may lead to biomechanical intervention of hepatocytes and the development of innovative tissue engineering for clinical treatment to target liver cells rather than exclusively focusing on the extracellular matrix alone in liver cirrhosis.

  18. In vitro culture of isolated primary hepatocytes and stem cell-derived hepatocyte-like cells for liver regeneration.

    PubMed

    Hu, Chenxia; Li, Lanjuan

    2015-08-01

    Various liver diseases result in terminal hepatic failure, and liver transplantation, cell transplantation and artificial liver support systems are emerging as effective therapies for severe hepatic disease. However, all of these treatments are limited by organ or cell resources, so developing a sufficient number of functional hepatocytes for liver regeneration is a priority. Liver regeneration is a complex process regulated by growth factors (GFs), cytokines, transcription factors (TFs), hormones, oxidative stress products, metabolic networks, and microRNA. It is well-known that the function of isolated primary hepatocytes is hard to maintain; when cultured in vitro, these cells readily undergo dedifferentiation, causing them to lose hepatocyte function. For this reason, most studies focus on inducing stem cells, such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), hepatic progenitor cells (HPCs), and mesenchymal stem cells (MSCs), to differentiate into hepatocyte-like cells (HLCs) in vitro. In this review, we mainly focus on the nature of the liver regeneration process and discuss how to maintain and enhance in vitro hepatic function of isolated primary hepatocytes or stem cell-derived HLCs for liver regeneration. In this way, hepatocytes or HLCs may be applied for clinical use for the treatment of terminal liver diseases and may prolong the survival time of patients in the near future.

  19. The role of prolactin and progesterone in the regulation of lipogenesis in maternal and foetal rat liver in vivo and in isolated hepatocytes during the last day of gestation.

    PubMed Central

    Lorenzo, M; Roncero, C; Benito, M

    1986-01-01

    The administration of progesterone on day 21 of gestation increases the rates of lipogenesis in the liver in vivo and in hepatocytes isolated from rats on day 22 of pregnancy. Bromocriptine administration increases the rates of hepatic lipogenesis in vivo, but has no effect on lipid synthesis in hepatocytes under the same treatment conditions. Concurrently, the administration of progesterone or bromocriptine on day 21 to the mother increases the rates of lipogenesis in the foetal liver in vivo on day 22. The rates of lipid synthesis in foetal isolated hepatocytes are increased by progesterone administration, but remain unchanged by bromocriptine. PMID:3800972

  20. Genomic characterization and dynamic methylation of promoter facilitates transcriptional regulation of H2A variants, H2A.1 and H2A.2 in various pathophysiological states of hepatocyte.

    PubMed

    Tyagi, Monica; Reddy, Divya; Gupta, Sanjay

    2017-02-03

    Differential expression of homomorphous variants of H2A family of histone H2A.1 and H2A.2 have been associated with hepatocellular carcinoma and maintenance of undifferentiated state of hepatocyte. However, not much is known about the transcriptional regulation of these H2A variants. The current study revealed the presence of 43bp 5'-regulatory region upstream of translation start site and a 26bp 3' stem loop conserved region for both the H2A.1 and H2A.2 variants. However, alignment of both H2A.1 and H2A.2 5'-untranslated region (UTR) sequences revealed no significant degree of homology between them despite the coding exon being very similar amongst the variants. Further, transient transfection coupled with dual luciferase assay of cloned 5' upstream sequences of H2A.1 and H2A.2 of length 1.2 (-1056 to +144) and 1.379kb (-1160 to +219) from experimentally identified 5'UTR in rat liver cell line (CL38) confirmed their promoter activity. Moreover, in silico analysis revealed a presence of multiple CpG sites interspersed in the cloned promoter of H2A.1 and a CpG island near TSS for H2A.2, suggesting that histone variants transcription might be regulated epigenetically. Indeed, treatment with DNMT and HDAC inhibitors increased the expression of H2A.2 with no significant change in H2A.1 levels. Further, methyl DNA immunoprecipitation coupled with quantitative analysis of DNA methylation using real-time PCR revealed hypo-methylation and hyper-methylation of H2A.1 and H2A.2 respectively in embryonic and HCC compared to control adult liver tissue. Collectively, the data suggests that differential DNA methylation on histone promoters is a dynamic player regulating their expression status in different pathophysiological stages of liver.

  1. Organic anion uptake by hepatocytes.

    PubMed

    Wolkoff, Allan W

    2014-10-01

    Many of the compounds taken up by the liver are organic anions that circulate tightly bound to protein carriers such as albumin. The fenestrated sinusoidal endothelium of the liver permits these compounds to have access to hepatocytes. Studies to characterize hepatic uptake of organic anions through kinetic analyses, suggested that it was carrier-mediated. Attempts to identify specific transporters by biochemical approaches were largely unsuccessful and were replaced by studies that utilized expression cloning. These studies led to identification of the organic anion transport proteins (oatps), a family of 12 transmembrane domain glycoproteins that have broad and often overlapping substrate specificities. The oatps mediate Na(+)-independent organic anion uptake. Other studies identified a seven transmembrane domain glycoprotein, Na(+)/taurocholate transporting protein (ntcp) as mediating Na(+)-dependent uptake of bile acids as well as other organic anions. Although mutations or deficiencies of specific members of the oatp family have been associated with transport abnormalities, there have been no such reports for ntcp, and its physiologic role remains to be determined, although expression of ntcp in vitro recapitulates the characteristics of Na(+)-dependent bile acid transport that is seen in vivo. Both ntcp and oatps traffic between the cell surface and intracellular vesicular pools. These vesicles move through the cell on microtubules, using the microtubule based motors dynein and kinesins. Factors that regulate this motility are under study and may provide a unique mechanism that can alter the plasma membrane content of these transporters and consequently their accessibility to circulating ligands.

  2. Response of MiRNA-22-3p and MiRNA-149-5p to Folate Deficiency and the Differential Regulation of MTHFR Expression in Normal and Cancerous Human Hepatocytes

    PubMed Central

    Li, Chao; Ni, Juan; Liu, Yao-Xian; Wang, Han; Liang, Zi-Qing; Wang, Xu

    2017-01-01

    Background/Aims Folic acid (FA) is a core micronutrient involved in DNA synthesis/methylation, and the metabolism of FA is responsible for genomic stability. MicroRNAs may affect gene expression during folate metabolism when cellular homeostasis is changed. This study aimed to reveal the relationship between FA deficiency and the expression of miR-22-p/miR-149-5p and the targeted regulation of miR-22-3p/miR-149-5p on the key folate metabolic gene Methylenetetrahydrofolate reductase (MTHFR). Methods Normal (HL-7702 cells) and cancerous (QGY-7703 cells) human hepatocytes were intervened in modified RPMI 1640 with FA deficiency for 21 days. The interaction between MTHFR and the tested miRNAs was verified by Dual-Luciferase Reporter Assays. The changes in the expression of miR-22-3p/miR-149-5p in response to FA deficiency were detected by Poly (A) Tailing RT-qPCR, and the expression of MTHFR at both the transcriptional and translational levels was determined by RT-qPCR and Western blotting, respectively. Result MiR-22-3p/miR-149-5p directly targeted the 3’UTR sequence of the MTHFR gene. FA deficiency led to an upregulation of miR-22-3p/miR-149-5p expression in QGY-7703/HL-7702 cells, while the transcription of MTHFR was decreased in QGY-7703 cells but elevated in HL-7702 cells. Western blotting showed that FA deficiency resulted in a decline of the MTHFR protein in QGY-7703 cells, whereas in HL-7702 cells, the MTHFR protein level remained constant. Conclusion The results suggested that miR-22-3p/miR-149-5p exert different post-transcriptional effects on MTHFR under conditions of FA deficiency in normal and cancerous human hepatocytes. The results also implied that miR-22-3p/miR-149-5p might exert anticancer effects in cases of long-term FA deficiency. PMID:28045918

  3. Voodoo in the general hospital. A case of hexing and regional enteritis.

    PubMed

    Cappannari, S C; Rau, B; Abram, H S; Buchanan, D C

    1975-06-02

    We have observed the case of a hexed patient that emphasizes the interplay between modern medical practice and the influence of a supernatural voodoo subculture. Psychosocial stresses involved in the patient's illness (regional enteritis) added to the complexity of the clinical problem. The presence of ambivalence in the patient and her mother concerning their systems of belief, and their unwillingness to accept totally the treatment modalities of either modern medicine or voodoo, probably prevented the patient's predicted death.

  4. Unsteady flow simulations in support of the SSME HEX turning vane cracking investigation with the ATD HPOTP

    NASA Astrophysics Data System (ADS)

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

    1993-07-01

    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

  5. [Cloning, expression and characterization of a new hybrid AMP gene of Hex-Mag].

    PubMed

    Li, Gui-ping; Chen, Yi-ben

    2007-02-01

    To enhance the antibacterial ability of Magaininl-12, its N side was joined with an alkaline peptide named Hexapeptide( RRWQWR), which would make Magaininl-12 cling to the membrane of bacterial cells even tighter. According to the partiality codon of Pichia pastoris, a new hybrid antibacterial peptide Hex-Mag was designed based on the sequence of Hexapeptide and Magainin( 1-12). Synthesized through gene splicing by overlap extension, the hybrid gene was cloned into pPIC9 to construct the expression vector pPIC9-HM. After restriction enzyme analysis and purification, the pPIC9-HM was transformed into Pichia pastoris GS115. And the positive clones screened by the phenotype were induced by methanol. After optimized the requirements for the flask-shaking culture fermentation, the hybrid antibacterial peptide was expressed on high level. The new peptide, which has a weight of 2.3kDa, could remain its inhibition activity after treating for more than 3 hours in boiled water. Detected by agrose diffusion assay, Hex-Mag showed its broad-spectrum antibacterial abilities not only to Gram-negative bacteria but also to Gram-positive bacteria. The function of additive positive charges were testified by the antibacterial experiments, and the results showed the activity of Hex-Mag was stronger than that of Magainin1-12 obviously.

  6. 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.

  7. Cancer Associated Fibroblast-Derived Hepatocyte Growth Factor Inhibits the Paclitaxel-Induced Apoptosis of Lung Cancer A549 Cells by Up-Regulating the PI3K/Akt and GRP78 Signaling on a Microfluidic Platform

    PubMed Central

    Xu, Zhiyun; He, Tianrui; Li, Encheng; Guo, Zhe; Liu, Fen; Jiang, Chunmeng; Wang, Qi

    2015-01-01

    Tumor stroma and growth factors provide a survival environment to tumor cells and can modulate their chemoresistance by dysregulating several signal pathways. In this study, we fabricated a three-dimensional (3D) microfluidic chip using polydimethylsiloxane (PDMS) to investigate the impact of hepatocyte growth factor (HGF) from cancer-associated fibroblasts (CAF) on the Met/PI3K/AKT activation, glucose regulatory protein (GRP78) expression and the paclitaxel-induced A549 cell apoptosis. With a concentration gradient generator, the assembled chip was able to reconstruct a tumor microenvironment in vitro. We found high levels of HGF in the supernatants of CAF and the CAF matrix from the supernatants of activated HFL1 fibroblasts or HGF enhanced the levels of Met, PI3K and AKT phosphorylation and GRP78 expression in A549 cells cultured in a 3D cell chamber, which was abrogated by anti-HGF. Inhibition of Met attenuated the CAF matrix-enhanced PI3K/AKT phosphorylation and GRP78 expression while inhibition of PI3K reduced GRP78 expression, but not Met phosphorylation in A549 cells. Inhibition of GRP78 failed to modulate the CAF matrix-enhanced Met/PI3K/AKT phosphorylation in A549 cells. Furthermore, inhibition of PI3K or GRP78 enhanced spontaneous and paclitaxel-induced A549 cell apoptosis. Moreover, treatment with the CAF matrix inhibited spontaneous and medium or high dose of paclitaxel-induced A549 cell apoptosis. Inhibition of PI3K or GRP78 attenuated the CAF matrix-mediated inhibition on paclitaxel-induced A549 cell apoptosis. Our data indicated that HGF in the CAF matrix activated the Met/PI3K/AKT and up-regulated GRP78 expression, promoting chemoresistance to paclitaxel-mediated apoptosis in A549 cells. Our findings suggest that the microfluidic system may represent an ideal platform for signaling research and drug screening. PMID:26115510

  8. The current status of primary hepatocyte culture

    PubMed Central

    Mitaka, Toshihiro

    1998-01-01

    Recently, there have been significant advances toward the development of culture conditions that promote proliferation of primary rodent hepatocytes. There are two major methods for the multiplication of hepatocytes in vitro: one is the use of nicotinamide, the other is the use of a nutrient-rich medium. In the medium containing a high concentration of nicotinamide and a growth factor, primary hepatocytes can proliferate well. In this culture condition small mononucleate cells, which are named small hepatocytes, appear and form colonies. Small hepatocytes have a high potential to proliferate while maintaining hepatic characteristics, and can differentiate into mature ones. On the other hand, combining the nutrient-rich medium with 2% DMSO, the proliferated hepatocytes can recover the hepatic differentiated functions and maintain them for a long time. In this review I describe the culture conditions for the proliferation and differentiation of primary hepatocytes and discuss the small hepatocytes, especially their roles in liver growth. PMID:10319020

  9. 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

  10. Autophagy: a cyto-protective mechanism which prevents primary human hepatocyte apoptosis during oxidative stress.

    PubMed

    Bhogal, Ricky H; Weston, Christopher J; Curbishley, Stuart M; Adams, David H; Afford, Simon C

    2012-04-01

    The role of autophagy in the response of human hepatocytes to oxidative stress remains unknown. Understanding this process may have important implications for the understanding of basic liver epithelial cell biology and the responses of hepatocytes during liver disease. To address this we isolated primary hepatocytes from human liver tissue and exposed them ex vivo to hypoxia and hypoxia-reoxygenation (H-R). We showed that oxidative stress increased hepatocyte autophagy in a reactive oxygen species (ROS) and class III PtdIns3K-dependent manner. Specifically, mitochondrial ROS and NADPH oxidase were found to be key regulators of autophagy. Autophagy involved the upregulation of BECN1, LC3A, Atg7, Atg5 and Atg 12 during hypoxia and H-R. Autophagy was seen to occur within the mitochondria of the hepatocyte and inhibition of autophagy resulted in the lowering a mitochondrial membrane potential and onset of cell death. Autophagic responses were primarily observed in the large peri-venular (PV) hepatocyte subpopulation. Inhibition of autophagy, using 3-methyladenine, increased apoptosis during H-R. Specifically, PV human hepatocytes were more susceptible to apoptosis after inhibition of autophagy. These findings show for the first time that during oxidative stress autophagy serves as a cell survival mechanism for primary human hepatocytes.

  11. Using reconfigurable microfluidics to study the role of HGF in autocrine and paracrine signaling of hepatocytes.

    PubMed

    Patel, Dipali; Haque, Amranul; Gao, Yandong; Revzin, Alexander

    2015-07-01

    Cancer, developmental biology and tissue injury present multiple examples where groups of cells residing in close proximity communicate via paracrine factors. It is nearly impossible to dissect such cellular interactions in vivo and is quite challenging in vitro. The goal of this study is to utilize a reconfigurable microfluidic device in order to study paracrine signal exchange between groups of primary hepatocytes in vitro. Previously, we demonstrated that hepatocytes residing on protein spots containing collagen and hepatocyte growth factor (HGF) spots expressed epithelial (hepatic) phenotypes and also rescued them in neighboring hepatocytes on collagen spots that did not receive direct HGF stimulus. Herein, we designed a microfluidic device with parallel fluidic channels separated by retractable (reconfigurable) walls and employed this device to investigate interactions between groups of HGF-stimulated and unstimulated hepatocytes. Using a novel reconfigurable microfluidic device, we demonstrate that cultivation of HGF-containing protein spots upregulates the production of endogenous HGF in hepatocytes and that these HGF molecules diffuse over, causing phenotype enhancement in the recipient cells. We also show that selective treatment of the recipient hepatocytes with a c-met inhibitor (SU11274) diminishes the rescue effect, as gauged by the down-regulation of albumin and HGF expression. Our study is one of the first to demonstrate paracrine signaling via HGF in primary hepatocytes. More broadly, tools and methods described here may be used to study paracrine signaling in other types of cells and will have relevance for various fields of biomedical research from cancer to immunology.

  12. Members of the nuclear factor 1 family and hepatocyte nuclear factor 4 bind to overlapping sequences of the L-II element on the rat pyruvate kinase L gene promoter and regulate its expression.

    PubMed

    Yamada, K; Tanaka, T; Noguchi, T

    1997-06-15

    The L-II element (-149 to -126 bp) in the enhancer unit of the rat pyruvate kinase L (PKL) gene is required for cell-type-specific transcription and induction by carbohydrates. This element was found to bind multiple nuclear proteins with different heat stabilities. A heat-labile factor was shown to be hepatocyte nuclear factor (HNF) 4 by the electrophoretic mobility-shift assay (EMSA) using various competitor DNAs and anti-HNF4 serum. A heat-stable factor was purified from rat liver nuclear extract and was resolved as two protein bands migrating at about 33 kDa on SDS/polyacrylamide gels. Peptide sequence analysis revealed that these proteins were nuclear factor (NF) 1-L and NF1/Red1. The heat-stable factor was also identified as a member of the NF1 family by using various competitor DNAs and anti-NF1 serum in an EMSA. In addition, we found that a factor bound to the accessory site of the rat S14 gene, which is necessary for carbohydrate responsiveness of this gene, was also a member of the NF1 family, raising the possibility that the NF1 family is involved in the carbohydrate regulation of gene transcription by interactions with other proteins. The NF1 family members and HNF4 interacted with overlapping sequences of the L-II element, wherein the 5' half-site was more critical for NF1 binding, and the 3' site was more important for HNF4 binding. Co-transfection of a vector expressing either NF1-L or NF1/Red1 repressed the transcription of the PKL enhancer unit-chloramphenicol acetyltransferase (CAT) fusion gene in HepG2 cells, whereas co-transfection of a vector expressing HNF4 activated the transcription of the same reporter gene. Furthermore NF1 family members antagonized the effect of HNF4 on PKL enhancer unit-CAT fusion gene expression when both expression plasmids were co-transfected. We conclude that NF1 family members and HNF4 regulate transcription of the PKL gene in an opposing manner by binding overlapping sequences of the L-II element.

  13. Restructuring of hex-Pt(100) under CO gas environments: formation of 2-D nanoclusters

    SciTech Connect

    Tao, Feng; Dag, Sefa; Wang, Lin-Wang; Liu, Zhi; Butcher, Derek; Salmeron, Miquel; Somorjai, Gabor A.

    2009-04-24

    The atomic-scale restructuring of hex-Pt(100) induced by carbon monoxide with a wide pressure range was studied with a newly designed chamber-in-chamber high-pressure STM and theoretical calculations. Both experimental and DFT calculation results show that CO molecules are bound to Pt nanoclusters through a tilted on-top configuration with a separation of {approx}3.7-4.1 {angstrom}. The phenomenon of restructuring of metal catalyst surfaces induced by adsorption, and in particular the formation of small metallic clusters suggests the importance of studying structures of catalyst surfaces under high pressure conditions for understanding catalytic mechanisms.

  14. 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.

  15. Marked increase in biofilm-derived rough pneumococcal variants and rifampin-resistant strains not due to hex gene mutations.

    PubMed

    McEllistrem, M Catherine; Scott, Jennifer R; Zuniga-Castillo, Jacobo; Khan, Saleem A

    2009-06-01

    Otitis, pneumonia, and meningitis are tissue-based pneumococcal infections that can be associated with biofilms. The emergence of phenotypic rough variants, also known as acapsular small-colony variants, is essential for pneumococcal biofilm formation. These rough variants can increase nearly 100-fold in biofilms over time and can arise through single nucleotide polymorphisms (SNPs), deletions, or tandem duplications in the first gene of the capsular operon, cps3D. We detected a 100-fold increase in rifampin-resistant (Rif(r)) mutants in biofilms compared to planktonic cultures using a nonvaccine serotype 3 strain, which is causing an increasing number of cases of otitis in the 7-valent pneumococcal conjugate vaccine era. Since both rough variants and Rif(r) strains can arise through SNPs, they could emerge due to alteration of the mismatch repair (MMR) system. The Hex system, a pneumococcal MMR system, repairs mismatches during replication and transformation. In this study, no mutations were detected in the hexAB gene sequences among several rough variants with unique mutations in the cps3D gene. Within a hexA null mutant grown in broth, we detected only a 17.5-fold increase in rough variants compared to the wild-type parental strain. Taken together, these data suggest that mutations in the hex genes and modulation of hexA activity are unlikely to account for the generation of biofilm-derived rough variants.

  16. 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

  17. Electrochemical sensing of hepatocyte viability.

    PubMed

    Tsai, Hweiyan; Tsai, Shang-heng; Ting, Wei-Jen; Hu, Chao-Chin; Fuh, C Bor

    2014-05-21

    We investigated the use of amperometric and chronoamperometric methods with a double mediator system and screen-printed electrodes (SPEs) for the electrochemical sensing of hepatocyte viability. Cell counts were determined based on measuring cellular respiration via interaction of electroactive redox mediators. The oxidation currents of chronoamperometric measurement were proportional to the concentrations of ferrocyanide which was produced via interaction of cellular respiration, succinate and ferricyanide. The integrated oxidation charges increased linearly with the density of the cultured primary rat hepatocytes over a range of 1 × 10(5) to 5 × 10(5) cells per well (slope = 1.98 (±0.08) μC per 10(5) cells; R(2) = 0.9969), and the detection limit was 7600 (±300) cells per well based on S/N = 3. Each density of cells was cultured in triple replicates and individual cell samples were evaluated. The results of the cytotoxic effect of the chronoamperometric method are comparable to those of the tetrazolium-based colorimetric assay. The chronoamperometric method with ferricyanide and succinate mediators is an efficient, alternative method for assessing the viability of primary hepatocytes which can be completed in 20 min. Succinate did not provide an efficient electron shuttle between cytosolic respiratory redox activity of cancer cells and extracellular ferricyanide, an effect that may be useful for distinguishing hepatocarcinoma cells from healthy hepatocytes.

  18. 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.

  19. 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.

  20. 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.

  1. Characterization of the major Woronin body protein HexA of the human pathogenic mold Aspergillus fumigatus.

    PubMed

    Beck, Julia; Ebel, Frank

    2013-03-01

    In filamentous fungi, the septal pore controls the exchange between neighbouring hyphal compartments. Woronin bodies are fungal-specific organelles that plug the pore in case of physical damage. The Hex protein is their major and essential component. Hex proteins of different size are predicted in the data base for pathogenic and non-pathogenic Aspergillus species. However, using specific monoclonal antibodies, we identified 2 dominant HexA protein species of 20 and 25kDa in A. fumigatus, A. terreus, A. nidulans, and A. oryzae. HexA and Woronin bodies were found in A. fumigatus hyphae, but also in resting conidia. Using monoclonal antibodies, a GFP-HexA fusion protein, and an RFP protein fused to the putative peroxisomal targeting sequence of HexA, we analyzed the spatial localization and dynamics of Woronin bodies in A. fumigatus as well as their formation from peroxisomes. In intact hyphae, some Woronin bodies were found in close proximity to the septal pore, while the majority was distributed in the cytoplasm. Septum-associated Woronin bodies show a minimal lateral movement, while the cytosolic Woronin bodies are highly dynamic. The distribution of Woronin bodies and their co-localization pattern with peroxisomes revealed no evidence that Woronin bodies arise predominantly at the apical tip of A. fumigatus hyphae. We found that Woronin bodies are able to plug septal pores of A. fumigatus in case of damage. Woronin bodies therefore contribute to the stress resistance and potentially also to the virulence of A. fumigatus, which renders them a potential target for future anti-fungal strategies.

  2. TGFbeta Induces Binucleation/Polyploidization in Hepatocytes through a Src-Dependent Cytokinesis Failure

    PubMed Central

    Grassi, Germana; Bisceglia, Francesca; Strippoli, Raffaele; Guarguaglini, Giulia; Citarella, Franca; Sacchetti, Benedetto; Tripodi, Marco; Amicone, Laura

    2016-01-01

    In all mammals, the adult liver shows binucleated as well as mononucleated polyploid hepatocytes. The hepatic polyploidization starts after birth with an extensive hepatocyte binucleation and generates hepatocytes of several ploidy classes. While the functional significance of hepatocyte polyploidy is becoming clearer, how it is triggered and maintained needs to be clarified. Aim of this study was to identify a major inducer of hepatocyte binucleation/polyploidization and the cellular and molecular mechanisms involved. We found that, among several cytokines analyzed, known to be involved in early liver development and/or mass control, TGFbeta1 was capable to induce, together with the expected morphological changes, binucleation in hepatocytes in culture. Most importantly, the pharmacological inhibition of TGFbeta signaling in healthy mice during weaning, when the physiological binucleation occurs, induced a significant decrease of hepatocyte binucleation rate, without affecting cell proliferation and hepatic index. The TGFbeta-induced hepatocyte binucleation resulted from a cytokinesis failure, as assessed by video microscopy, and is associated with a delocalization of the cytokinesis regulator RhoA-GTPase from the mid-body of dividing cells. The use of specific chemical inhibitors demonstrated that the observed events are Src-dependent. Finally, the restoration of a fully epithelial phenotype by TGFbeta withdrawal gave rise to a cell progeny capable to maintain the polyploid state. In conclusion, we identified TGFbeta as a major inducer of hepatocyte binucleation both in vitro and in vivo, thus ascribing a novel role to this pleiotropic cytokine. The production of binucleated/tetraploid hepatocytes is due to a cytokinesis failure controlled by the molecular axis TGFbeta/Src/RhoA. PMID:27893804

  3. Enhanced Cell Survival and Yield of Rat Small Hepatocytes by Honeycomb-Patterned Films

    NASA Astrophysics Data System (ADS)

    Tsukiyama, Shusaku; Matsushita, Michiaki; Tanaka, Masaru; Tamura, Hitoshi; Todo, Satoru; Yamamoto, Sadaaki; Shimomura, Masatsugu

    2008-02-01

    Surface designing of substrate to regulate cell adhesion and function in nano and micro scale is a critical issue in biomaterial science. In this study, we describe the fabrication of highly regular patterned porous films (honeycomb-patterned film) formed by a simply casting technique, and the culture of mature hepatocytes and small hepatocytes on the films. The pore size of the honeycomb-patterned films used was 6, 12, and 16 µm. We evaluated the effect of the honeycomb-patterned films on the morphology, cell yield, survival and the differentiated hepatic function (albumin production) of the both hepatocytes. Both hepatocytes attached on the flat films appeared to spread well, showing a typical monolayer morphology. They peeled off from the films at 7 days in culture on the flat films. On the other hand, spreading of the each hepatocytes was restricted on the honeycomb-patterned films at 3 and 7 days in culture. The cell yield and survival of the each hepatocytes increased with increasing culture time. Small hepatocyte on the pore sizes of 16 µm showed the highest cell yield (approximately 3 times). Albumin production of mature hepatocyte on the pore sizes of 16 µm (224.1.3 ±157 ng ml-1 well-1 at 1 day in culture, 369.5 ±222 ng ml-1 well-1 at 3 days in culture) was higher than that of the hepatocytes on the flat films (119.3 ±9.3 ng ml-1 well-1 at 1 day in culture, 262.8 ±47.3 ng ml-1 well-1 at 3 days in culture), although that of small hepatocytes on the honeycomb-patterned films (pore size: 16 µm) was similar on the flat film. These results indicated that both the surface topography and the pore size of the honeycomb-patterned film affected the hepatic metabolic function.

  4. Toxin-Induced Activation of Rat Hepatocyte Prostaglandin Synthesis and Phospholipid Metabolism

    DTIC Science & Technology

    1989-12-22

    SUBJECT TERMS (Continue on reverse if necesay and identify by block number) FIELD GROUP SUB-GROUP - microcystin -LR, arachidonic acid, phospholipid...pool was reduced to 47% (p ɘ.025) by l’ 3’ microcystin -LR. Changes in phospholipid classes indicated that prostaglandin formation induced by microcystin ... microcystin -LR has important effects on the regulation of inflammatory mediator synthesis in hepatocytes. 7.,1 ,- TOXIN-INDUCED ACTIVATION OF RAT HEPATOCYTE

  5. Hepatitis B virus modulates store-operated calcium entry to enhance viral replication in primary hepatocytes

    PubMed Central

    Casciano, Jessica C.; Duchemin, Nicholas J.; Lamontagne, R. Jason; Steel, Laura F.; Bouchard, Michael J.

    2017-01-01

    Many viruses modulate calcium (Ca2+) signaling to create a cellular environment that is more permissive to viral replication, but for most viruses that regulate Ca2+ signaling, the mechanism underlying this regulation is not well understood. The hepatitis B virus (HBV) HBx protein modulates cytosolic Ca2+ levels to stimulate HBV replication in some liver cell lines. A chronic HBV infection is associated with life-threatening liver diseases, including hepatocellular carcinoma (HCC), and HBx modulation of cytosolic Ca2+ levels could have an important role in HBV pathogenesis. Whether HBx affects cytosolic Ca2+ in a normal hepatocyte, the natural site of an HBV infection, has not been addressed. Here, we report that HBx alters cytosolic Ca2+ signaling in cultured primary hepatocytes. We used single cell Ca2+ imaging of cultured primary rat hepatocytes to demonstrate that HBx elevates the cytosolic Ca2+ level in hepatocytes following an IP3-linked Ca2+ response; HBx effects were similar when expressed alone or in the context of replicating HBV. HBx elevation of the cytosolic Ca2+ level required extracellular Ca2+ influx and store-operated Ca2+ (SOC) entry and stimulated HBV replication in hepatocytes. We used both targeted RT-qPCR and transcriptome-wide RNAseq analyses to compare levels of SOC channel components and other Ca2+ signaling regulators in HBV-expressing and control hepatocytes and show that the transcript levels of these various proteins are not affected by HBV. We also show that HBx regulation of SOC-regulated Ca2+ accumulation is likely the consequence of HBV modulation of a SOC channel regulatory mechanism. In support of this, we link HBx enhancement of SOC-regulated Ca2+ accumulation to Ca2+ uptake by mitochondria and demonstrate that HBx stimulates mitochondrial Ca2+ uptake in primary hepatocytes. The results of our study may provide insights into viral mechanisms that affect Ca2+ signaling to regulate viral replication and virus-associated diseases

  6. Hepatitis B virus modulates store-operated calcium entry to enhance viral replication in primary hepatocytes.

    PubMed

    Casciano, Jessica C; Duchemin, Nicholas J; Lamontagne, R Jason; Steel, Laura F; Bouchard, Michael J

    2017-01-01

    Many viruses modulate calcium (Ca2+) signaling to create a cellular environment that is more permissive to viral replication, but for most viruses that regulate Ca2+ signaling, the mechanism underlying this regulation is not well understood. The hepatitis B virus (HBV) HBx protein modulates cytosolic Ca2+ levels to stimulate HBV replication in some liver cell lines. A chronic HBV infection is associated with life-threatening liver diseases, including hepatocellular carcinoma (HCC), and HBx modulation of cytosolic Ca2+ levels could have an important role in HBV pathogenesis. Whether HBx affects cytosolic Ca2+ in a normal hepatocyte, the natural site of an HBV infection, has not been addressed. Here, we report that HBx alters cytosolic Ca2+ signaling in cultured primary hepatocytes. We used single cell Ca2+ imaging of cultured primary rat hepatocytes to demonstrate that HBx elevates the cytosolic Ca2+ level in hepatocytes following an IP3-linked Ca2+ response; HBx effects were similar when expressed alone or in the context of replicating HBV. HBx elevation of the cytosolic Ca2+ level required extracellular Ca2+ influx and store-operated Ca2+ (SOC) entry and stimulated HBV replication in hepatocytes. We used both targeted RT-qPCR and transcriptome-wide RNAseq analyses to compare levels of SOC channel components and other Ca2+ signaling regulators in HBV-expressing and control hepatocytes and show that the transcript levels of these various proteins are not affected by HBV. We also show that HBx regulation of SOC-regulated Ca2+ accumulation is likely the consequence of HBV modulation of a SOC channel regulatory mechanism. In support of this, we link HBx enhancement of SOC-regulated Ca2+ accumulation to Ca2+ uptake by mitochondria and demonstrate that HBx stimulates mitochondrial Ca2+ uptake in primary hepatocytes. The results of our study may provide insights into viral mechanisms that affect Ca2+ signaling to regulate viral replication and virus-associated diseases.

  7. 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.

  8. 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.

  9. Nrf2 is essential for timely M phase entry of replicating hepatocytes during liver regeneration

    PubMed Central

    Zou, Yuhong; Hu, Min; Lee, Joonyong; Nambiar, Shashank Manohar; Garcia, Veronica; Bao, Qi; Chan, Jefferson Y.

    2014-01-01

    The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates various cellular activities, including redox balance, detoxification, metabolism, autophagy, proliferation, and apoptosis. Several studies have demonstrated that Nrf2 regulates hepatocyte proliferation during liver regeneration. The aim of this study was to investigate how Nrf2 modulates the cell cycle of replicating hepatocytes in regenerating livers. Wild-type and Nrf2 null mice were subjected to 2/3 partial hepatectomy (PH) and killed at multiple time points for various analyses. Nrf2 null mice exhibited delayed liver regrowth, although the lost liver mass was eventually restored 7 days after PH. Nrf2 deficiency did not affect the number of hepatocytes entering the cell cycle but did delay hepatocyte mitosis. Mechanistically, the lack of Nrf2 resulted in increased mRNA and protein levels of hepatic cyclin A2 when the remaining hepatocytes were replicating in response to PH. Moreover, Nrf2 deficiency in regenerating livers caused dysregulation of Wee1, Cdc2, and cyclin B1 mRNA and protein expression, leading to decreased Cdc2 activity. Thus, Nrf2 is required for timely M phase entry of replicating hepatocytes by ensuring proper regulation of cyclin A2 and the Wee1/Cdc2/cyclin B1 pathway during liver regeneration. PMID:25524062

  10. Hepatocyte nuclear factor 4alpha controls the development of a hepatic epithelium and liver morphogenesis.

    PubMed

    Parviz, Fereshteh; Matullo, Christine; Garrison, Wendy D; Savatski, Laura; Adamson, John W; Ning, Gang; Kaestner, Klaus H; Rossi, Jennifer M; Zaret, Kenneth S; Duncan, Stephen A

    2003-07-01

    Although advances have been made in understanding cell differentiation, only rudimentary knowledge exists concerning how differentiated cells form tissues and organs. We studied liver organogenesis because the cell and tissue architecture of this organ is well defined. Approximately 60% of the adult liver consists of hepatocytes that are arranged as single-cell anastomosing plates extending from the portal region of the liver lobule toward the central vein. The basal surface of the hepatocytes is separated from adjacent sinusoidal endothelial cells by the space of Disse, where the exchange of substances between serum and hepatocytes takes place. The hepatocyte's apical surface forms bile canaliculi that transport bile to the hepatic ducts. Proper liver architecture is crucial for hepatic function and is commonly disrupted in disease states, including cirrhosis and hepatitis. Here we report that hepatocyte nuclear factor 4alpha (Hnf4alpha) is essential for morphological and functional differentiation of hepatocytes, accumulation of hepatic glycogen stores and generation of a hepatic epithelium. We show that Hnf4alpha is a dominant regulator of the epithelial phenotype because its ectopic expression in fibroblasts induces a mesenchymal-to-epithelial transition. Most importantly, the morphogenetic parameters controlled by Hnf4alpha in hepatocytes are essential for normal liver architecture, including the organization of the sinusoidal endothelium.

  11. Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes.

    PubMed

    Akie, Thomas E; Cooper, Marcus P

    2015-08-27

    Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders. In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via β-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying β-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of β-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

  12. On hexenuronic acid (HexA) removal and mediator coupling to pulp fiber in the laccase/mediator treatment.

    PubMed

    Cadena, Edith M; Du, Xueyu; Gellerstedt, Göran; Li, Jiebing; Fillat, Amanda; García-Ubasart, Jordi; Vidal, Teresa; Colom, Josep F

    2011-02-01

    Flax soda/AQ pulps were treated with different fungal laccase-mediator combinations followed by physical and chemical characterization of the pulps to obtain a thorough understanding of the laccase/mediator effects on hexenuronic acid (HexA) removal and the coupling of mediator onto pulps for fiber functionalization. Large differences were found and the presence of lauryl gallate (LG) during Trametes villosa laccase (TvL) treatment (TvL+LG) resulted in a much larger reduction of pulp-linked HexA than the combination of p-coumaric acid (PCA) and Pycnoporus cinnabarinus laccase (PcL). A major portion of LG became attached to the pulp as revealed by an increase in the kappa number and further confirmed by thioacidolysis and (1)H NMR analysis of solubilized pulp fractions. Additional experiments with other chemical pulps and isolated pulp xylan and lignin revealed that HexA seems to be the sole pulp component attacked by TvL+LG. As a substrate for TvL, the reaction preference order is PCA>HexA>LG.

  13. HexSim: A flexible simulation model for forecasting wildlife responses to multiple interacting stressors - ESRP Meeting

    EPA Science Inventory

    With SERDP funding, we have improved upon a popular life history simulator (PATCH), and indoing 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 life...

  14. Immediate loading of titanium hexed screw-type implants in the edentulous patient: case report.

    PubMed

    Calvo, M P; Muller, E; Garg, A K

    2000-01-01

    Histologic and histomorphometric studies in both animals and humans have shown that more rapid and greater bone-to-implant contact can be achieved with implants that incorporate certain surface characteristics compared with the original machined-surface implants. Such findings are significant because various implant designs may allow the fixtures to sufficiently resist functional loading sooner than originally thought. The case report presented here indicates that immediate loading of hexed titanium screw-type implants in the anterior mandible can lead to successful osseointegration and clinical outcome. The number of implants placed, their distribution, and the type of rigid connection are critical considerations for immediate loading. A bone height that can accommodate dental implants > or = 10 mm long is recommended. Biomechanically, the implants to be immediately loaded must be stable and resistant to macromovement to ensure good osseointegration.

  15. HexServer: an FFT-based protein docking server powered by graphics processors.

    PubMed

    Macindoe, Gary; Mavridis, Lazaros; Venkatraman, Vishwesh; Devignes, Marie-Dominique; Ritchie, David W

    2010-07-01

    HexServer (http://hexserver.loria.fr/) is the first Fourier transform (FFT)-based protein docking server to be powered by graphics processors. Using two graphics processors simultaneously, a typical 6D docking run takes approximately 15 s, which is up to two orders of magnitude faster than conventional FFT-based docking approaches using comparable resolution and scoring functions. The server requires two protein structures in PDB format to be uploaded, and it produces a ranked list of up to 1000 docking predictions. Knowledge of one or both protein binding sites may be used to focus and shorten the calculation when such information is available. The first 20 predictions may be accessed individually, and a single file of all predicted orientations may be downloaded as a compressed multi-model PDB file. The server is publicly available and does not require any registration or identification by the user.

  16. Robust rat pulmonary radioprotection by a lipophilic Mn N-alkylpyridylporphyrin, MnTnHex-2-PyP(5+).

    PubMed

    Gauter-Fleckenstein, Benjamin; Reboucas, Julio S; Fleckenstein, Katharina; Tovmasyan, Artak; Owzar, Kouros; Jiang, Chen; Batinic-Haberle, Ines; Vujaskovic, Zeljko

    2014-01-01

    With the goal to enhance the distribution of cationic Mn porphyrins within mitochondria, the lipophilic Mn(III)meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin, MnTnHex-2-PyP(5+) has been synthesized and tested in several different model of diseases, where it shows remarkable efficacy at as low as 50 µg/kg single or multiple doses. Yet, in a rat lung radioprotection study, at higher 0.6-1 mg/kg doses, due to its high accumulation and micellar character, it became toxic. To avoid the toxicity, herein the pulmonary radioprotection of MnTnHex-2-PyP(5+) was assessed at 50 µg/kg. Fischer rats were irradiated to their right hemithorax (28 Gy) and treated with 0.05 mg/kg/day of MnTnHex-2-PyP(5+) for 2 weeks by subcutaneously-implanted osmotic pumps, starting at 2 h post-radiation. The body weights and breathing frequencies were followed for 10 weeks post-radiation, when the histopathology and immunohistochemistry were assessed. Impact of MnTnHex-2-PyP(5+) on macrophage recruitment (ED-1), DNA oxidative damage (8-OHdG), TGF-β1, VEGF(A) and HIF-1α were measured. MnTnHex-2-PyP(5+) significantly decreased radiation-induced lung histopathological (H&E staining) and functional damage (breathing frequencies), suppressed oxidative stress directly (8-OHdG), or indirectly, affecting TGF-β1, VEGF (A) and HIF-1α pathways. The magnitude of the therapeutic effects is similar to the effects demonstrated under same experimental conditions with 120-fold higher dose of ~5000-fold less lipophilic Mn(III)meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin, MnTE-2-PyP(5+).

  17. 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

  18. 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.

  19. 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

  20. Hepatocyte Death: A Clear and Present Danger

    PubMed Central

    MALHI, HARMEET; GUICCIARDI, MARIA EUGENIA; GORES, GREGORY J.

    2010-01-01

    The hepatocyte is especially vulnerable to injury due to its central role in xenobiotic metabolism including drugs and alcohol, participation in lipid and fatty acid metabolism, its unique role in the enterohepatic circulation of bile acids, the widespread prevalence of hepatotropic viruses, and its existence within a milieu of innate immune responding cells. Apoptosis and necrosis are the most widely recognized forms of hepatocyte cell death. The hepatocyte displays many unique features regarding cell death by apoptosis. It is quite susceptible to death receptor-mediated injury, and its death receptor signaling pathways involve the mitochondrial pathway for efficient cell killing. Also, death receptors can trigger lysosomal disruption in hepatocytes which further promote cell and tissue injury. Interestingly, hepatocytes are protected from cell death by only two anti-apoptotic proteins, Bcl-xL and Mcl-1, which have nonredundant functions. Endoplasmic reticulum stress or the unfolded protein response contributes to hepatocyte cell death during alterations of lipid and fatty acid metabolism. Finally, the current information implicating RIP kinases in necrosis provides an approach to more fully address this mode of cell death in hepatocyte injury. All of these processes contributing to hepatocyte injury are discussed in the context of potential therapeutic strategies. PMID:20664081

  1. Regulation of Glucose Metabolism in Pseudomonas

    PubMed Central

    Daddaoua, Abdelali; Krell, Tino; Ramos, Juan-Luis

    2009-01-01

    In Pseudomonas putida, genes for the glucose phosphorylative pathway and the Entner-Doudoroff pathway are organized in two operons; one made up of the zwf, pgl, and eda genes and another consisting of the edd, glk, gltR2, and gltS genes. Divergently with respect to the edd gene is the gap-1 gene. Expression from Pzwf, Pedd, and Pgap is modulated by HexR in response to the availability of glucose in the medium. To study the regulatory process in greater detail we purified HexR and showed that it is a monomer in solution. Electrophoretic mobility shift assays and isothermal titration calorimetry assays were done showing that HexR recognizes the Pedd, Pzwf, and Pgap-1 promoters with affinity in the nanomolar range. DNA footprinting assays identified the binding site between +30 and +1 at Pzwf, between +16 and +41 at Pedd, and between −6 and +18 at Pgap-1. Based on DNA sequence alignment of the target sites and isothermal titration calorimetry data, two monomers of HexR bind to a pseudopalindrome with a consensus sequence of 5′-TTGTN7–8ACAA-3′. Binding of the Entner-Doudoroff pathway intermediate 2-keto-3-deoxy-6-phosphogluconate to HexR released the repressor from its target operators, whereas other chemicals such as glucose, glucose 6-phosphate, and 6-phosphogluconate did not induce complex dissociation. The phosphorylated effector is likely to be recognized by a sugar isomerase domain located at the C-terminal end of HexR, whereas the helix-turn-helix DNA binding domain of HexR exhibits high similarity to proteins of the RpiR family of regulators. PMID:19506074

  2. Longitudinal in vivo bioimaging of hepatocyte transcription factor activity following cholestatic liver injury in mice

    PubMed Central

    Delhove, Juliette M. K. M.; Buckley, Suzanne M. K.; Perocheau, Dany P.; Karda, Rajvinder; Arbuthnot, Patrick; Henderson, Neil C.; Waddington, Simon N.; McKay, Tristan R.

    2017-01-01

    Molecular mechanisms regulating liver repair following cholestatic injury remain largely unknown. We have combined a mouse model of acute cholestatic liver injury, partial bile duct ligation (pBDL), with a novel longitudinal bioimaging methodology to quantify transcription factor activity during hepatic injury and repair. We administered lentiviral transcription factor activated luciferase/eGFP reporter (TFAR) cassettes to neonatal mice enabling longitudinal TFAR profiling by continued bioimaging throughout the lives of the animals and following pBDL in adulthood. Neonatal intravascular injection of VSV-G pseudotyped lentivirus resulted in almost exclusive transduction of hepatocytes allowing analysis of hepatocyte-specific transcription factor activity. We recorded acute but transient responses with NF-κB and Smad2/3 TFAR whilst our Notch reporter was repressed over the 40 days of evaluation post-pBDL. The bipotent hepatic progenitor cell line, HepaRG, can be directed to differentiate into hepatocytes and biliary epithelia. We found that forced expression of the Notch inhibitor NUMB in HepaRG resulted in enhanced hepatocyte differentiation and proliferation whereas over-expressing the Notch agonist JAG1 resulted in biliary epithelial differentiation. In conclusion, our data demonstrates that hepatocytes rapidly upregulate NF-κB and Smad2/3 activity, whilst repressing Notch signalling. This transcriptional response to cholestatic liver injury likely promotes partial de-differentiation to allow pro-regenerative proliferation of hepatocytes. PMID:28157201

  3. 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.

  4. YAP inhibition restores hepatocyte differentiation in advanced HCC leading to tumor regression

    PubMed Central

    Fitamant, Julien; Kottakis, Filippos; Benhamouche, Samira; Tian, Helen S.; Chuvin, Nicolas; Parachoniak, Christine A.; Nagle, Julia M.; Perera, Rushika M.; Lapouge, Marjorie; Deshpande, Vikram; Zhu, Andrew X.; Lai, Albert; Min, Bosun; Hoshida, Yujin; Avruch, Joseph; Sia, Daniela; Campreciós, Genís; McClatchey, Andrea I.; Llovet, Josep M.; Morrissey, David; Raj, Lakshmi; Bardeesy, Nabeel

    2015-01-01

    SUMMARY Defective Hippo/YAP signaling in the liver results in tissue overgrowth and development of hepatocellular carcinoma (HCC). Here, we uncover mechanisms of YAP-mediated hepatocyte reprogramming and HCC pathogenesis. YAP functions as a rheostat maintaining metabolic specialization, differentiation and quiescence within the hepatocyte compartment. Increased or decreased YAP activity reprograms subsets of hepatocytes to different fates associated with deregulation of the HNF4A, CTNNB1, and E2F transcriptional programs controlling hepatocyte quiescence and differentiation. Importantly, treatment with siRNA-lipid nanoparticles (siRNA-LNPs) targeting YAP restores hepatocyte differentiation and causes pronounced tumor regression in a genetically engineered mouse HCC model. Furthermore, YAP targets are enriched in an aggressive human HCC subtype characterized by a proliferative signature and absence of CTNNB1 mutations. Thus, our work reveals Hippo signaling as a key regulator of positional identity of hepatocytes, supports targeting YAP using siRNA-LNPs as a paradigm of differentiation-based therapy, and identifies an HCC subtype potentially responsive to this approach. PMID:25772357

  5. Longitudinal in vivo bioimaging of hepatocyte transcription factor activity following cholestatic liver injury in mice.

    PubMed

    Delhove, Juliette M K M; Buckley, Suzanne M K; Perocheau, Dany P; Karda, Rajvinder; Arbuthnot, Patrick; Henderson, Neil C; Waddington, Simon N; McKay, Tristan R

    2017-02-03

    Molecular mechanisms regulating liver repair following cholestatic injury remain largely unknown. We have combined a mouse model of acute cholestatic liver injury, partial bile duct ligation (pBDL), with a novel longitudinal bioimaging methodology to quantify transcription factor activity during hepatic injury and repair. We administered lentiviral transcription factor activated luciferase/eGFP reporter (TFAR) cassettes to neonatal mice enabling longitudinal TFAR profiling by continued bioimaging throughout the lives of the animals and following pBDL in adulthood. Neonatal intravascular injection of VSV-G pseudotyped lentivirus resulted in almost exclusive transduction of hepatocytes allowing analysis of hepatocyte-specific transcription factor activity. We recorded acute but transient responses with NF-κB and Smad2/3 TFAR whilst our Notch reporter was repressed over the 40 days of evaluation post-pBDL. The bipotent hepatic progenitor cell line, HepaRG, can be directed to differentiate into hepatocytes and biliary epithelia. We found that forced expression of the Notch inhibitor NUMB in HepaRG resulted in enhanced hepatocyte differentiation and proliferation whereas over-expressing the Notch agonist JAG1 resulted in biliary epithelial differentiation. In conclusion, our data demonstrates that hepatocytes rapidly upregulate NF-κB and Smad2/3 activity, whilst repressing Notch signalling. This transcriptional response to cholestatic liver injury likely promotes partial de-differentiation to allow pro-regenerative proliferation of hepatocytes.

  6. Hepatocyte transplantation-biology and application.

    PubMed

    Gewartowska, M; Olszewski, Waldemar L

    2007-01-01

    Transplantation of liver has been remarkably effective in the treatment of liver failure and liver-based inherited metabolic diseases and has revolutionized the care of patients with end-stage liver disease. Unfortunately demand for transplantable livers is progressively outpacing the supply of donated cadaver organs, resulting in longer waiting times and increased mortality for prospective transplant recipients. Hepatocyte transplantation has been proposed as a method to support patients with liver insufficiency. The current knowledge on this method has been review in this article. Now the two-step collagenase perfusion technique is widely used for isolation of hepatocytes. Liver has been considered as an optimal site for hepatocyte transplantation, however, even in this organ the survival rate of transplanted hepatocytes in extremely low. The main obstacle for wider usage of hepatocyte transplantation is their rapid elimination by recipient macrophages. We tried in animal experiments to downregulate the innate process of recipient cellular attack on implanted hepatocytes by irradiation of recipient and elimination of NK cells. Ligation of bile duct and partial hepatectomies facilitated proliferation of accepted donor hepatocytes and formation of bile canaliculi. The described method is now adjusted to clinical conditions.

  7. 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.

  8. Platform switching on wide-diameter external hex implants: a finite element analysis

    PubMed Central

    Sivolella, Stefano; Bressan, Eriberto; Berengo, Mario; Stellini, Edoardo

    2013-01-01

    Objectives: The objective of this work was to use finite element analysis to compare the effect of forces coming to bear on abutments 4.1 or 5.0 mm in diameter connected to a 5.0 mm implant (i.e. with or without platform switching). Study design: A 3D CAD model of a 5 x 11.5 mm external hex implant was developed, complete with a connection screw and either of two abutments, one 4.1 and the other 5 mm in diameter, to assess the influence of two loading conditions, i.e. 200 N loaded either axially or off center on the top of the abutment. Results and conclusions: In the symmetrically loaded models, greater stresses were transmitted to the bone in the area below the neck of the implant in the case of the wider-diameter abutment. When the narrower abutment was considered, the stress lines remained confined to the metal and were transferred to the bone in a more distal position. When the stresses in the bone where compared under non-symmetrical loading of the larger- and smaller-diameter abutments, the stresses reached lower values in the latter case. These findings indicate that platform switching (i.e. coupling a 4.1 mm abutment with a 5 mm implant) achieves a better, more even distribution of the peri-implant stresses deriving from simulated occlusal loads on the bone margins. Key words:Platform switching, finite element analysis, implant. PMID:24455061

  9. Progressive induction of hepatocyte progenitor cells in chronically injured liver

    PubMed Central

    Tanimizu, Naoki; Ichinohe, Norihisa; Yamamoto, Masahiro; Akiyama, Haruhiko; Nishikawa, Yuji; Mitaka, Toshihiro

    2017-01-01

    Differentiated epithelial cells show substantial lineage plasticity upon severe tissue injuries. In chronically injured mouse livers, part of hepatocytes become Sry-HMG box containing 9 (Sox9) (+) epithelial cell adhesion molecule (−) hepatocyte nuclear factor 4 α (+) biphenotypic hepatocytes. However, it is not clear whether all Sox9+ hepatocytes uniformly possess cellular properties as hepatocyte progenitors. Here, we examined the microarray data comparing Sox9+ hepatocytes with mature hepatocytes and identified CD24 as a novel marker for biphenotypic hepatocytes. Immunohistochemical analyses showed that part of Sox9+ hepatocytes near expanded ductular structures expressed CD24 in the liver injured by 3,5-diethoxycarbonyl-1,4-dihydro-collidine (DDC) diet and by bile duct ligation. Indeed, Sox9+ hepatocytes could be separated into CD24− and CD24+ cells by fluorescence activated cell sorting. The ratio of CD24+ cells against CD24− ones in Sox9+ hepatocytes gradually increased while DDC-injury progressed and colony-forming capability mostly attributed to CD24+ cells. Although hepatocyte markers were remarkably downregulated in of Sox9+ CD24+ hepatocytes, they re-differentiated into mature hepatocytes in vitro and in vivo. Our current results demonstrate that the emergence of biphenotypic hepatocytes is a sequential event including the transition from CD24− and CD24+ status, which may be a crucial step for hepatocytes to acquire progenitor properties. PMID:28051157

  10. 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.

  11. Sequence and stress-response analyses of the DNA mismatch repair gene hexA in Lactococcus lactis.

    PubMed

    Ren, J; Park, J H; Dunn, N W; Kim, W S

    2001-10-01

    The DNA mismatch repair gene hexA was identified in Lactococcus lactis by PCR amplification by using a pair of primers homologous to the DNA-binding Dps protein. The gene in its entirety, including the regulatory regions, was sequenced, by using a strategy of chromosomal walking based on two PCR protocols. The open reading frame of 2526 bp was preceded by a strong ribosome-binding site (AGGAAG) and was followed by a potential transcription terminator (hairpin loop structure). The 5' terminus of the hexA mRNA was located 135 bp upstream of the start codon, and putative -10 and -35 regions were identified. The deduced amino acid sequence revealed two motifs, the ATP/GTP-binding site (P-loop) and the "MutS family signature". The hexA promoter was cloned into pMU1327, which contained a promoter-less CAT reporter gene, and the promoter activity was examined under oxidative-stress conditions. It appears that the promoter activity is down-shifted by H2O2 at 4 mM.

  12. 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.

  13. 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

  14. In utero Transplanted Human Hepatocytes Allows for Postnatal Engraftment of Human Hepatocytes in Pigs

    PubMed Central

    Fisher, James E; Lillegard, Joseph B; Mckenzie, Travis J; Rodysill, Brian R; Wettstein, Peter J; Nyberg, Scott L

    2012-01-01

    In utero cell transplantation (IUCT) can lead to postnatal engraftment of human cells in the xenogeneic recipient. Most reports of IUCTs have involved hematopoietic stem cells. It is unknown if human hepatocytes used for IUCT in fetal pigs will lead to engraftment of these same cells in the postnatal environment. In this study, fetal pigs received direct liver injections of 1×107 human hepatocytes in utero and were delivered by cesarean-section at term. Piglets received a second direct liver injection of 5×107 human hepatocytes 1 week postnatally. Serum was analyzed for human albumin at 2, 4, and 6 weeks post-engraftment. Piglet livers were harvested 6 weeks after transplantation and examined by immunohistochemistry, PCR and fluorescence in situ hybridization for human specific sequences. Piglets receiving IUCT with human hepatocytes that were postnatally engrafted with human hepatocytes showed significant levels of human albumin production in their serum at all post-engraftment time points. Human albumin gene expression, the presence of human hepatocytes and the presence of human beta-2 microglobulin were all confirmed 6 weeks post-engraftment. IUCT in fetal pigs using human hepatocytes early in gestation allowed for engraftment of human hepatocytes, which remained viable and functional for weeks after transplantation. IUCT followed by postnatal engraftment may provide a future means for large scale expansion of human hepatocytes in genetically-engineered pigs. PMID:23280879

  15. 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

  16. 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.

  17. Reduced expression of mature TGF beta 1 correlates with the suppression of rat hepatocyte apoptosis by the peroxisome proliferator, nafenopin.

    PubMed

    Strange, J; Roberts, R A

    1996-11-11

    Non-genotoxic carcinogens cause cancer without damaging the DNA. Peroxisome proliferators (PPs) are a class of potent rodent non-genotoxic hepatocarcinogens that may act by perturbing hepatocyte growth regulation. Previously, we have shown that although cultured rat hepatocytes degenerate rapidly in culture, their survival can be reversibly maintained by the PP nafenopin. This prolonged survival is associated with a decrease in the number of hepatocytes displaying the chromatin condensation characteristic of apoptosis. The addition of the negative growth regulator TGF beta-1 induced high levels of hepatocyte apoptosis but nafenopin was able to suppress this TGF beta-1 induced apoptosis. These data suggested that increased levels of mature TGF beta-1 may be involved in the signalling of the apoptosis seen in degenerating hepatocyte cultures. To test this hypothesis, we carried out Western blot analyses using a anti-TGF beta 1 antibody. There was an increase (p = 0.014) in expression of mature TGF beta 1 in degenerating rat hepatocyte cultures compared with hepatocyte cultures surviving in the presence of nafenopin. However, there was a concomitant decrease (p = 0.024) in TGF beta 1-latency activated protein (TGF beta 1-LAP), the precursor of the active, mature form. Immunocytochemistry confirmed that TGF beta 1/TGF beta 1-LAP expression was predominantly in the hepatocytes displaying apoptotic morphology although expression was detected also in non-parenchymal liver cells. The immunocytochemistry data indicate that TGF beta 1 is involved during the onset of hepatocyte apoptosis and that the PP nafenopin can impinge on this cell death pathway. TGF beta 1-LAP, probably produced mainly by the non-parenchymal liver cells, may be processed less efficiently to the mature, active form in the presence of nafenopin, although more data are required to confirm this hypothesis.

  18. 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.

  19. Role of hepatocyte nuclear factor 4α (HNF4α) in cell proliferation and cancer.

    PubMed

    Walesky, Chad; Apte, Udayan

    2015-01-01

    Hepatocyte nuclear factor 4α (HNF4α) is an orphan nuclear receptor commonly known as the master regulator of hepatic differentiation, owing to the large number of hepatocyte-specific genes it regulates. Whereas the role of HNF4α in hepatocyte differentiation is well recognized and extensively studied, its role in regulation of cell proliferation is relatively less known. Recent studies have revealed that HNF4α inhibits proliferation not only of hepatocytes but also cells in colon and kidney. Further, a growing number of studies have demonstrated that inhibition or loss of HNF4α promotes tumorigenesis in the liver and colon, and reexpression of HNF4α results in decreased cancer growth. Studies using tissue-specific conditional knockout mice, knock-in studies, and combinatorial bioinformatics of RNA/ChIP-sequencing data indicate that the mechanisms of HNF4α-mediated inhibition of cell proliferation are multifold, involving epigenetic repression of promitogenic genes, significant cross talk with other cell cycle regulators including c-Myc and cyclin D1, and regulation of miRNAs. Furthermore, studies indicate that posttranslational modifications of HNF4α may change its activity and may be at the core of its dual role as a differentiation factor and repressor of proliferation. This review summarizes recent findings on the role of HNF4α in cell proliferation and highlights the newly understood function of this old receptor.

  20. 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.

  1. 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.

  2. 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

    2015-05-14

    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.

  3. Nrf2 Knockdown Disrupts the Protective Effect of Curcumin on Alcohol-Induced Hepatocyte Necroptosis.

    PubMed

    Lu, Chunfeng; Xu, Wenxuan; Zhang, Feng; Shao, Jiangjuan; Zheng, Shizhong

    2016-12-05

    It has emerged that hepatocyte necroptosis plays a critical role in chronic alcoholic liver disease (ALD). Our previous study has identified that the beneficial therapeutic effect of curcumin on alcohol-caused liver injury might be attributed to activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), whereas the role of curcumin in regulating necroptosis and the underlying mechanism remain to be determined. We first found that chronic alcohol consumption triggered obvious hepatocyte necroptosis, leading to increased expression of receptor-interacting protein 1, receptor-interacting protein 3, high-mobility group box 1, and phosphorylated mixed lineage kinase domain-like in murine livers. Curcumin dose-dependently ameliorated hepatocyte necroptosis and alleviated alcohol-caused decrease in hepatic Nrf2 expression in alcoholic mice. Then Nrf2 shRNA lentivirus was introduced to generate Nrf2-knockdown mice. Our results indicated that Nrf2 knockdown aggravated the effects of alcohol on liver injury and necroptosis and even abrogated the inhibitory effect of curcumin on necroptosis. Further, activated Nrf2 by curcumin inhibited p53 expression in both livers and cultured hepatocytes under alcohol stimulation. The next in vitro experiments, similar to in vivo ones, revealed that although Nrf2 knockdown abolished the suppression of curcumin on necroptosis of hepatocytes exposed to ethanol, p53 siRNA could clearly rescued the relative effect of curcumin. In summary, for the first time, we concluded that curcumin attenuated alcohol-induced hepatocyte necroptosis in a Nrf2/p53-dependent mechanism. These findings make curcumin an excellent candidate for ALD treatment and advance the understanding of ALD mechanisms associated with hepatocyte necroptosis.

  4. Differentiation-Promoting Medium Additives for Hepatocyte Cultivation and Cryopreservation.

    PubMed

    Gouliarmou, Varvara; Pelkonen, Olavi; Coecke, Sandra

    2015-01-01

    Isolated primary hepatocytes are considered as the reference system for in vitro hepatic methods. Following the isolation of primary hepatocytes from liver tissue, an unfavorable process named dedifferentiation is initiated leading to the attenuation of the hepatocellular phenotype both at the morphological and functional level. Freshly isolated hepatocytes can be used immediately or can be cryopreserved for future purposes. Currently, a number of antidedifferentiation strategies exist to extend the life span of isolated hepatocytes. The addition of differentiation-promoting compounds to the hepatocyte culture medium is the oldest and simplest antidedifferentiation approach applied. In the present chapter, the most commonly used medium additives for cultivation and cryopreservation of primary hepatocytes are reviewed.

  5. 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

  6. Gene networks and transcription factor motifs defining the differentiation of stem cells into hepatocyte-like cells

    PubMed Central

    Godoy, Patricio; Schmidt-Heck, Wolfgang; Natarajan, Karthick; Lucendo-Villarin, Baltasar; Szkolnicka, Dagmara; Asplund, Annika; Björquist, Petter; Widera, Agata; Stöber, Regina; Campos, Gisela; Hammad, Seddik; Sachinidis, Agapios; Chaudhari, Umesh; Damm, Georg; Weiss, Thomas S.; Nüssler, Andreas; Synnergren, Jane; Edlund, Karolina; Küppers-Munther, Barbara; Hay, David C.; Hengstler, Jan G.

    2015-01-01

    Background & Aims The differentiation of stem cells to hepatocyte-like cells (HLC) offers the perspective of unlimited supply of human hepatocytes. However, the degree of differentiation of HLC remains controversial. To obtain an unbiased characterization, we performed a transcriptomic study with HLC derived from human embryonic and induced stem cells (ESC, hiPSC) from three different laboratories. Methods Genome-wide gene expression profiles of ESC and HLC were compared to freshly isolated and up to 14 days cultivated primary human hepatocytes. Gene networks representing successful and failed hepatocyte differentiation, and the transcription factors involved in their regulation were identified. Results Gene regulatory network analysis demonstrated that HLC represent a mixed cell type with features of liver, intestine, fibroblast and stem cells. The “unwanted” intestinal features were associated with KLF5 and CDX2 transcriptional networks. Cluster analysis identified highly correlated groups of genes associated with mature liver functions (n = 1057) and downregulated proliferation associated genes (n = 1562) that approach levels of primary hepatocytes. However, three further clusters containing 447, 101, and 505 genes failed to reach levels of hepatocytes. Key TF of two of these clusters include SOX11, FOXQ1, and YBX3. The third unsuccessful cluster, controlled by HNF1, CAR, FXR, and PXR, strongly overlaps with genes repressed in cultivated hepatocytes compared to freshly isolated hepatocytes, suggesting that current in vitro conditions lack stimuli required to maintain gene expression in hepatocytes, which consequently also explains a corresponding deficiency of HLC. Conclusions The present gene regulatory network approach identifies key transcription factors which require modulation to improve HLC differentiation. PMID:26022688

  7. Evaluation of stability of interface between CCM (Co-Cr-Mo) UCLA abutment and external hex implant

    PubMed Central

    Yoon, Ki-Joon; Park, Young-Bum; Choi, Hyunmin; Cho, Youngsung; Lee, Jae-Hoon

    2016-01-01

    PURPOSE The purpose of this study is to evaluate the stability of interface between Co-Cr-Mo (CCM) UCLA abutment and external hex implant. MATERIALS AND METHODS Sixteen external hex implant fixtures were assigned to two groups (CCM and Gold group) and were embedded in molds using clear acrylic resin. Screw-retained prostheses were constructed using CCM UCLA abutment and Gold UCLA abutment. The external implant fixture and screw-retained prostheses were connected using abutment screws. After the abutments were tightened to 30 Ncm torque, 5 kg thermocyclic functional loading was applied by chewing simulator. A target of 1.0 × 106 cycles was applied. After cyclic loading, removal torque values were recorded using a driving torque tester, and the interface between implant fixture and abutment was evaluated by scanning electronic microscope (SEM). The means and standard deviations (SD) between the CCM and Gold groups were analyzed with independent t-test at the significance level of 0.05. RESULTS Fractures of crowns, abutments, abutment screws, and fixtures and loosening of abutment screws were not observed after thermocyclic loading. There were no statistically significant differences at the recorded removal torque values between CCM and Gold groups (P>.05). SEM analysis revealed that remarkable wear patterns were observed at the abutment interface only for Gold UCLA abutments. Those patterns were not observed for other specimens. CONCLUSION Within the limit of this study, CCM UCLA abutment has no statistically significant difference in the stability of interface with external hex implant, compared with Gold UCLA abutment. PMID:28018564

  8. FGF19-induced hepatocyte proliferation is mediated through FGFR4 activation.

    PubMed

    Wu, Xinle; Ge, Hongfei; Lemon, Bryan; Vonderfecht, Steven; Weiszmann, Jennifer; Hecht, Randy; Gupte, Jamila; Hager, Todd; Wang, Zhulun; Lindberg, Richard; Li, Yang

    2010-02-19

    FGF19 and FGF21, unique members of the fibroblast growth factor (FGF) family, are hormones that regulate glucose, lipid, and energy homeostasis. Increased hepatocyte proliferation and liver tumor formation have also been observed in FGF19 transgenic mice. Here, we report that, in contrast to FGF19, FGF21 does not induce hepatocyte proliferation in vivo. To identify the mechanism for FGF19-induced hepatocyte proliferation, we explored similarities and differences in receptor specificity between FGF19 and FGF21. We find that although both are able to activate FGF receptors (FGFRs) 1c, 2c, and 3c, only FGF19 activates FGFR4, the predominant receptor in the liver. Using a C-terminal truncation mutant of FGF19 and a series of FGF19/FGF21 chimeric molecules, we determined that amino acids residues 38-42 of FGF19 are sufficient to confer both FGFR4 activation and increased hepatocyte proliferation in vivo to FGF21. These data suggest that activation of FGFR4 is the mechanism whereby FGF19 can increase hepatocyte proliferation and induce hepatocellular carcinoma formation.

  9. 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.

  10. 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

  11. 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

  12. Proliferation of rat small hepatocytes requires follistatin expression.

    PubMed

    Ooe, Hidekazu; Chen, Qijie; Kon, Junko; Sasaki, Kazunori; Miyoshi, Hiroyuki; Ichinohe, Norihisa; Tanimizu, Naoki; Mitaka, Toshihiro

    2012-06-01

    Small hepatocytes (SHs) are a subpopulation of hepatocytes that have high growth potential in culture and can differentiate into mature hepatocytes (MHs). The activin (Act)/follistatin (Fst) system critically contributes to homeostasis of cell growth in the normal liver. ActA and ActB consist of two disulfide-linked Inhibin (Inh)β subunits, InhβA and InhβB, respectively. Fst binds to Act and blocks its bioactivity. In the present study we carried out the experiments to clarify how Fst regulates the proliferation of SHs. The gene expression was analyzed using DNA microarray analysis, reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR, and protein expression was examined by western blots, immunocytochemistry, and enzyme-linked immunosorbent assay. RT-PCR showed that Fst expression was high in SHs and low in MHs. Although the ActA expression was opposite to that of Fst, ActB expression was high in SHs and low in MHs and increased with time in culture. Fst protein was detected in the cytoplasm of SHs and secreted into the culture medium. ActB protein was also secreted into the medium. Although the exogenous administration of ActA and ActB apparently suppressed the proliferation of SHs, apoptosis of SHs was not induced by treatment with ActA or ActB. On the other hand, Fst treatment did not affect the colony formation of SHs but prevented the inhibitory effect of ActA. Neutralization by the anti-Fst antibody resulted in the suppression of DNA synthesis in SHs, and small hairpin RNA against Fst suppressed the expansion of SH colonies. In conclusion, Fst expression is necessary for the proliferation of SHs.

  13. Second- and Third-Order Elastic Constants of Filaments of HexTow® IM7 Carbon Fiber

    NASA Astrophysics Data System (ADS)

    Oliveira, L.; Hitchcock, D.; Behlow, H.; Podila, R.; Skove, M. J.; Serkiz, S. M.; Rao, A. M.

    2014-03-01

    Single filaments of HexTow® IM7-12K carbon fiber were subjected to tensile measurements on a device which applies a known stress σ, and measures the resulting strain ɛ, and the change in resistivity Δρ. Young's modulus E, the resistivity ρ, the piezoresistivity Δρ/ρɛ, and the nonlinearity in the stress-strain relation δ, were determined to be 264.1 ± 16.0 GPa, 1.5 ± 0.1 × 10-3 Ω cm, 1.3 ± 0.1, and -4.96 ± 0.23, respectively. The values obtained for Young's modulus and the resistivity of the fiber are in reasonable agreement with the values reported by the manufacturer. To the best of our knowledge, this is the first report of a measurement of a third-order elastic constant of a single filament of HexTow® IM7-12K. Given the high elastic strains attainable in these fibers and the negative value of δ, the usual calculation of E from a linear fit to the stress-strain data leads to an incorrect higher value of E. According to the accepted thermodynamic definition of the elastic constants, one must use the initial slope of the stress-strain curve to evaluate E. We also observed that the glue used to secure the fiber has an influence on the apparent modulus of the fiber.

  14. O 2 interaction with Pt{100}-hex-R0.7°: scattering, sticking and saturating

    NASA Astrophysics Data System (ADS)

    Guo, X.-C.; Bradley, J. M.; Hopkinson, A.; King, D. A.

    1994-05-01

    The interaction of oxygen with the Pt{100}-hex-R0.7° surface has been studied using supersonic molecular beams at incident translational energies from 0.06 to 0.9 eV and surface temperatures from 300 to 600 K. Scattering measurements show the existence of both intrinsic and extrinsic precursor states, and the trapping probability into these states is high at low incident energies. However, sticking probability measurements on the clean surface indicate that O 2 dissociative adsorption on Pt{100}-hex-R0.7° is a direct activated process, in contrast to that on Pt{100}-(1 × 1) or Pt{111}. Strong temperature enhancement of the initial sticking probability has been observed and accounted for partly by a dynamical barrier model. The sticking probability varies strongly with oxygen coverage, which is explained through computer simulations of island formation. The formation of small islands is demonstrated by TEAS measurements. Thermal desorption measurements show that, at high incident energies above 0.5 eV, new states are populated and higher coverages, up to a full monolayer, are reached.

  15. The ornithine requirement of urea synthesis. Formation of ornithine from glutamine in hepatocytes.

    PubMed Central

    Lund, P; Wiggins, D

    1986-01-01

    In hepatocytes, urea synthesis from glutamine is independent of added ornithine, even when rates are high after stimulation of glutamine metabolism by dibutyryl cyclic AMP, phenylephrine or vasopressin. Incubation with glutamine increases tissue [ornithine]. The increases parallel those of [N-acetylglutamate] under different conditions. The ornithine requirement of urea synthesis increases with increasing supply of ammonia. A function of the unique, highly regulated, glutaminase of liver may be to regulate ornithine synthesis. PMID:3030272

  16. Gene expression analysis of primary normal human hepatocytes infected with human hepatitis B virus

    PubMed Central

    Ryu, Hyun Mi; Park, Sung Gyoo; Yea, Sung Su; Jang, Won Hee; Yang, Young-Il; Jung, Guhung

    2006-01-01

    AIM: To find the relationship between hepatitis B virus (HBV) and hepatocytes during the initial state of infection by cDNA microarray. METHODS: Primary normal human hepatocytes (PNHHs) were isolated and infected with HBV. From the PNHHs, RNA was isolated and inverted into complement DNA (cDNA) with Cy3- or Cy5- labeled dUTP for microarray analysis. The labeled cDNA was hybridized with microarray chip, including 4224 cDNAs. From the image of the microarray, expression profiles were produced and some of them were confirmed by RT-PCR, immunoblot analysis, and NF-κB luciferase reporter assay. RESULTS: From the cDNA microarray, we obtained 98 differentially regulated genes. Of the 98 genes, 53 were up regulated and 45 down regulated. Interestingly, in the up regulated genes, we found the TNF signaling pathway-related genes: LT-α, TRAF2, and NIK. By using RT-PCR, we confirmed the up-regulation of these genes in HepG2, Huh7, and Chang liver cells, which were transfected with pHBV1.2×, a plasmid encoding all HBV messages. Moreover, these three genes participated in HBV-mediated NF-κB activation. CONCLUSION: During the initial state of HBV infection, hepatocytes facilitate the activation of NF-κB through up regulation of LT-α, TRAF2, and NIK. PMID:16937494

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

    PubMed

    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.

  18. 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

  19. Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice.

    PubMed

    Czaya, Brian; Singh, Saurav; Yanucil, Christopher; Schramm, Karla; Faul, Christian; Grabner, Alexander

    2017-03-10

    The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

  20. Farnesoid X Receptor Inhibits the Transcriptional Activity of Carbohydrate Response Element Binding Protein in Human Hepatocytes

    PubMed Central

    Caron, Sandrine; Huaman Samanez, Carolina; Dehondt, Hélène; Ploton, Maheul; Briand, Olivier; Lien, Fleur; Dorchies, Emilie; Dumont, Julie; Postic, Catherine; Cariou, Bertrand; Lefebvre, Philippe

    2013-01-01

    The glucose-activated transcription factor carbohydrate response element binding protein (ChREBP) induces the expression of hepatic glycolytic and lipogenic genes. The farnesoid X receptor (FXR) is a nuclear bile acid receptor controlling bile acid, lipid, and glucose homeostasis. FXR negatively regulates hepatic glycolysis and lipogenesis in mouse liver. The aim of this study was to determine whether FXR regulates the transcriptional activity of ChREBP in human hepatocytes and to unravel the underlying molecular mechanisms. Agonist-activated FXR inhibits glucose-induced transcription of several glycolytic genes, including the liver-type pyruvate kinase gene (L-PK), in the immortalized human hepatocyte (IHH) and HepaRG cell lines. This inhibition requires the L4L3 region of the L-PK promoter, known to bind the transcription factors ChREBP and hepatocyte nuclear factor 4α (HNF4α). FXR interacts directly with ChREBP and HNF4α proteins. Analysis of the protein complex bound to the L4L3 region reveals the presence of ChREBP, HNF4α, FXR, and the transcriptional coactivators p300 and CBP at high glucose concentrations. FXR activation does not affect either FXR or HNF4α binding to the L4L3 region but does result in the concomitant release of ChREBP, p300, and CBP and in the recruitment of the transcriptional corepressor SMRT. Thus, FXR transrepresses the expression of genes involved in glycolysis in human hepatocytes. PMID:23530060

  1. Cyclin D1 inhibits hepatic lipogenesis via repression of carbohydrate response element binding protein and hepatocyte nuclear factor 4α.

    PubMed

    Hanse, Eric A; Mashek, Douglas G; Becker, Jennifer R; Solmonson, Ashley D; Mullany, Lisa K; Mashek, Mara T; Towle, Howard C; Chau, Anhtung T; Albrecht, Jeffrey H

    2012-07-15

    Following acute hepatic injury, the metabolic capacity of the liver is altered during the process of compensatory hepatocyte proliferation by undefined mechanisms. In this study, we examined the regulation of de novo lipogenesis by cyclin D1, a key mediator of hepatocyte cell cycle progression. In primary hepatocytes, cyclin D1 significantly impaired lipogenesis in response to glucose stimulation. Cyclin D1 inhibited the glucose-mediated induction of key lipogenic genes, and similar effects were seen using a mutant (D1-KE) that does not activate cdk4 or induce cell cycle progression. Cyclin D1 (but not D1-KE) inhibited the activity of the carbohydrate response element-binding protein (ChREBP) by regulating the glucose-sensing motif of this transcription factor. Because changes in ChREBP activity could not fully explain the effect of cyclin D1, we examined hepatocyte nuclear factor 4α (HNF4α), which regulates numerous differentiated functions in the liver including lipid metabolism. We found that both cyclins D1 and D1-KE bound to HNF4α and significantly inhibited its recruitment to the promoter region of lipogenic genes in hepatocytes. Conversely, knockdown of cyclin D1 in the AML12 hepatocyte cell line promoted HNF4α activity and lipogenesis. In mouse liver, HNF4α bound to a central domain of cyclin D1 involved in transcriptional repression. Cyclin D1 inhibited lipogenic gene expression in the liver following carbohydrate feeding. Similar findings were observed in the setting of physiologic cyclin D1 expression in the regenerating liver. In conclusion, these studies demonstrate that cyclin D1 represses ChREBP and HNF4α function in hepatocytes via Cdk4-dependent and -independent mechanisms. These findings provide a direct link between the cell cycle machinery and the transcriptional control of metabolic function of the liver.

  2. WNT5A Inhibits Hepatocyte Proliferation and Concludes β-Catenin Signaling in Liver Regeneration

    PubMed Central

    Yang, Jing; Cusimano, Antonella; Monga, Jappmann K.; Preziosi, Morgan E.; Pullara, Filippo; Calero, Guillermo; Lang, Richard; Yamaguchi, Terry P.; Nejak-Bowen, Kari N.; Monga, Satdarshan P.

    2016-01-01

    Activation of Wnt/β-catenin signaling during liver regeneration (LR) after partial hepatectomy (PH) is observed in several species. However, how this pathway is turned off when hepatocyte proliferation is no longer required is unknown. We assessed LR in liver-specific knockouts of Wntless (Wls-LKO), a protein required for Wnt secretion from a cell. When subjected to PH, Wls-LKO showed prolongation of hepatocyte proliferation for up to 4 days compared with littermate controls. This coincided with increased β-catenin–T-cell factor 4 interaction and cyclin-D1 expression. Wls-LKO showed decreased expression and secretion of inhibitory Wnt5a during LR. Wnt5a expression increased between 24 and 48 hours, and Frizzled-2 between 24 and 72 hours, after PH in normal mice. Treatment of primary mouse hepatocytes and liver tumor cells with Wnt5a led to a notable decrease in β-catenin–T-cell factor activity, cyclin-D1 expression, and cell proliferation. Intriguingly, Wnt5a-LKO did not display any prolongation of LR because of compensation by other cells. In addition, Wnt5a-LKO hepatocytes failed to respond to exogenous Wnt5a treatment in culture because of a compensatory decrease in Frizzled-2 expression. In conclusion, we demonstrate Wnt5a to be, by default, a negative regulator of β-catenin signaling and hepatocyte proliferation, both in vitro and in vivo. We also provide evidence that the Wnt5a/Frizzled-2 axis suppresses β-catenin signaling in hepatocytes in an autocrine manner, thereby contributing to timely conclusion of the LR process. PMID:26100214

  3. WNT5A inhibits hepatocyte proliferation and concludes β-catenin signaling in liver regeneration.

    PubMed

    Yang, Jing; Cusimano, Antonella; Monga, Jappmann K; Preziosi, Morgan E; Pullara, Filippo; Calero, Guillermo; Lang, Richard; Yamaguchi, Terry P; Nejak-Bowen, Kari N; Monga, Satdarshan P

    2015-08-01

    Activation of Wnt/β-catenin signaling during liver regeneration (LR) after partial hepatectomy (PH) is observed in several species. However, how this pathway is turned off when hepatocyte proliferation is no longer required is unknown. We assessed LR in liver-specific knockouts of Wntless (Wls-LKO), a protein required for Wnt secretion from a cell. When subjected to PH, Wls-LKO showed prolongation of hepatocyte proliferation for up to 4 days compared with littermate controls. This coincided with increased β-catenin-T-cell factor 4 interaction and cyclin-D1 expression. Wls-LKO showed decreased expression and secretion of inhibitory Wnt5a during LR. Wnt5a expression increased between 24 and 48 hours, and Frizzled-2 between 24 and 72 hours, after PH in normal mice. Treatment of primary mouse hepatocytes and liver tumor cells with Wnt5a led to a notable decrease in β-catenin-T-cell factor activity, cyclin-D1 expression, and cell proliferation. Intriguingly, Wnt5a-LKO did not display any prolongation of LR because of compensation by other cells. In addition, Wnt5a-LKO hepatocytes failed to respond to exogenous Wnt5a treatment in culture because of a compensatory decrease in Frizzled-2 expression. In conclusion, we demonstrate Wnt5a to be, by default, a negative regulator of β-catenin signaling and hepatocyte proliferation, both in vitro and in vivo. We also provide evidence that the Wnt5a/Frizzled-2 axis suppresses β-catenin signaling in hepatocytes in an autocrine manner, thereby contributing to timely conclusion of the LR process.

  4. 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.

  5. Targeted transplantation of mitochondria to hepatocytes

    PubMed Central

    Gupta, Nidhi; Wu, Catherine H; Wu, George Y

    2016-01-01

    Background Mitochondrial defects in hepatocytes can result in liver dysfunction and death. Hepatocytes have cell-surface asialoglycoprotein receptors (AsGRs) which internalize AsGs within endosomes. The aim of this study was to determine whether mitochondria could be targeted to hepatocytes by AsGR-mediated endocytosis. Materials and methods An AsG, AsOR, was linked to polylysine to create a conjugate, AsOR-PL, and complexed with healthy and functional mitochondria (defined by normal morphology, cytochrome c assays, and oxygen-consumption rates). Huh7 (AsGR+) and SK Hep1 (AsGR−) cells were treated with a mitochondrial toxin to form Huh7-Mito− and SK Hep1-Mito− cells, lacking detectable mitochondrial DNA. An endosomolytic peptide, LLO, was coupled to AsOR to form AsOR-LLO. A lysosomal inhibitor, amantadine, was used in mitochondria-uptake studies as a control for nonspecific endosomal release. Results Coincubation of complexed mitochondria and AsOR-LLO with Huh7-Mito− cells increased mitochondrial DNA to >9,700-fold over control at 7 days (P<0.001), and increased mitochondrial oxygen-consumption rates to >90% of control by 10 days. Conclusion Rescue of mitochondria-damaged hepatocytes can be achieved by targeted uptake of normal mitochondria through receptor-mediated endocytosis. PMID:27942238

  6. Hypoxia Signaling Cascade for Erythropoietin Production in Hepatocytes.

    PubMed

    Tojo, Yutaka; Sekine, Hiroki; Hirano, Ikuo; Pan, Xiaoqing; Souma, Tomokazu; Tsujita, Tadayuki; Kawaguchi, Shin-ichi; Takeda, Norihiko; Takeda, Kotaro; Fong, Guo-Hua; Dan, Takashi; Ichinose, Masakazu; Miyata, Toshio; Yamamoto, Masayuki; Suzuki, Norio

    2015-08-01

    Erythropoietin (Epo) is produced in the kidney and liver in a hypoxia-inducible manner via the activation of hypoxia-inducible transcription factors (HIFs) to maintain oxygen homeostasis. Accelerating Epo production in hepatocytes is one plausible therapeutic strategy for treating anemia caused by kidney diseases. To elucidate the regulatory mechanisms of hepatic Epo production, we analyzed mouse lines harboring liver-specific deletions of genes encoding HIF-prolyl-hydroxylase isoforms (PHD1, PHD2, and PHD3) that mediate the inactivation of HIF1α and HIF2α under normal oxygen conditions. The loss of all PHD isoforms results in both polycythemia, which is caused by Epo overproduction, and fatty livers. We found that deleting any combination of two PHD isoforms induces polycythemia without steatosis complications, whereas the deletion of a single isoform induces no apparent phenotype. Polycythemia is prevented by the loss of either HIF2α or the hepatocyte-specific Epo gene enhancer (EpoHE). Chromatin analyses show that the histones around EpoHE dissociate from the nucleosome structure after HIF2α activation. HIF2α also induces the expression of HIF3α, which is involved in the attenuation of Epo production. These results demonstrate that the total amount of PHD activity is more important than the specific function of each isoform for hepatic Epo expression regulated by a PHD-HIF2α-EpoHE cascade in vivo.

  7. Methamphetamine enhances Hepatitis C virus replication in human hepatocytes.

    PubMed

    Ye, L; Peng, J S; Wang, X; Wang, Y J; Luo, G X; Ho, W Z

    2008-04-01

    Very little is known about the interactions between hepatitis C virus (HCV) and methamphetamine, which is a highly abused psychostimulant and a known risk factor for human immunodeficiency virus (HIV)/HCV infection. This study examined whether methamphetamine has the ability to inhibit innate immunity in the host cells, facilitating HCV replication in human hepatocytes. Methamphetamine inhibited intracellular interferon alpha expression in human hepatocytes, which was associated with the increase in HCV replication. In addition, methamphetamine also compromised the anti-HCV effect of recombinant interferon alpha. Further investigation of mechanism(s) responsible for the methamphetamine action revealed that methamphetamine was able to inhibit the expression of the signal transducer and activator of transcription 1, a key modulator in interferon-mediated immune and biological responses. Methamphetamine also down-regulated the expression of interferon regulatory factor-5, a crucial transcriptional factor that activates the interferon pathway. These in vitro findings that methamphetamine compromises interferon alpha-mediated innate immunity against HCV infection indicate that methamphetamine may have a cofactor role in the immunopathogenesis of HCV disease.

  8. Nontransferrin-bound iron uptake by hepatocytes is increased in the Hfe knockout mouse model of hereditary hemochromatosis.

    PubMed

    Chua, Anita C G; Olynyk, John K; Leedman, Peter J; Trinder, Debbie

    2004-09-01

    Hereditary hemochromatosis (HH) is an iron-overload disorder caused by a C282Y mutation in the HFE gene. In HH, plasma nontransferrin-bound iron (NTBI) levels are increased and NTBI is bound mainly by citrate. The aim of this study was to examine the importance of NTBI in the pathogenesis of hepatic iron loading in Hfe knockout mice. Plasma NTBI levels were increased 2.5-fold in Hfe knockout mice compared with control mice. Total ferric citrate uptake by hepatocytes isolated from Hfe knockout mice (34.1 +/- 2.8 pmol Fe/mg protein/min) increased by 2-fold compared with control mice (17.8 +/- 2.7 pmol Fe/mg protein/min; P <.001; mean +/- SEM; n = 7). Ferrous ion chelators, bathophenanthroline disulfonate, and 2',2-bipyridine inhibited ferric citrate uptake by hepatocytes from both mouse types. Divalent metal ions inhibited ferric citrate uptake by hepatocytes, as did diferric transferrin. Divalent metal transporter 1 (DMT1) mRNA and protein expression was increased approximately 2-fold by hepatocytes from Hfe knockout mice. We conclude that NTBI uptake by hepatocytes from Hfe knockout mice contributed to hepatic iron loading. Ferric ion was reduced to ferrous ion and taken up by hepatocytes by a pathway shared with diferric transferrin. Inhibition of uptake by divalent metals and up-regulation of DMT1 expression suggested that NTBI uptake was mediated by DMT1.

  9. 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.

  10. PTP1B deficiency increases glucose uptake in neonatal hepatocytes: involvement of IRA/GLUT2 complexes.

    PubMed

    González-Rodriguez, Agueda; Nevado, Carmen; Escrivá, Fernando; Sesti, Giorgio; Rondinone, Cristina M; Benito, Manuel; Valverde, Angela M

    2008-08-01

    The contribution of the liver to glucose utilization is essential to maintain glucose homeostasis. Previous data from protein tyrosine phosphatase (PTP) 1B-deficient mice demonstrated that the liver is a major site for PTP1B action in the periphery. In this study, we have investigated the consequences of PTP1B deficiency in glucose uptake in hepatocytes from neonatal and adult mice. The lack of PTP1B increased basal glucose uptake in hepatocytes from neonatal (3-5 days old) but not adult (10-12 wk old) mice. This occurs without changes in hexokinase, glucokinase, and glucose 6-phosphatase enzymatic activities. By contrast, the glucose transporter GLUT2 was upregulated at the protein level in neonatal hepatocytes and livers from PTP1B-deficient neonates. These results were accompanied by a significant increase in the net free intrahepatic glucose levels in the livers of PTP1B(-/-) neonates. The association between GLUT2 and insulin receptor (IR) A isoform was increased in PTP1B(-/-) neonatal hepatocytes compared with the wild-type. Indeed, PTP1B deficiency in neonatal hepatocytes shifted the ratio of isoforms A and B of the IR by increasing the amount of IRA and decreasing IRB. Moreover, overexpression of IRA in PTP1B(-/-) neonatal hepatocytes increased the amount of IRA/GLUT2 complexes. Conversely, hepatocytes from adult mice only expressed IRB. Since IRA plays a direct role in the regulation of glucose uptake in neonatal hepatocytes through its specific association with GLUT2, we propose the increase in IRA/GLUT2 complexes due to PTP1B deficiency as the molecular mechanism of the increased glucose uptake in the neonatal stage.

  11. Cryopreservation of primarily isolated porcine hepatocytes with UW solution.

    PubMed

    Kunieda, Takemi; Maruyama, Masanobu; Okitsu, Teru; Shibata, Norikuni; Takesue, Michihiko; Totsugawa, Toshinori; Kosaka, Yoshikazu; Arata, Takashi; Kobayashi, Kazuya; Ikeda, Hideaki; Oshita, Mizuko; Nakaji, Shuhei; Ohmoto, Kenji; Yamamoto, Shinichiro; Kurabayashi, Yuzuru; Kodama, Makoto; Tanaka, Noriaki; Kobayashi, Naoya

    2003-01-01

    Development of liver-targeted cell therapies, such as hepatocyte transplantation and bioartificial livers, requires a large amount of functional hepatocytes as needed. To achieve this development, establishing an excellent cryopreservation method of hepatocytes is an extremely important issue. Therefore, we performed a comparative review of cryoprotective effects of various cryopreservation solutions using primarily isolated porcine hepatocytes. Porcine hepatocytes were isolated with a four-step dispase and collagenase perfusion method. The obtained hepatocytes with the initial viabilities of 76%, 84%, and 96% were assigned to the following four groups for cryopreservation at -80 degrees C: Dulbecco's modified Eagle's medium (DMEM) + 10% fetal bovine serum (FBS) + 12% dimethyl sulfoxide (DMSO) (group A), University of Wisconsin (UW) solution + 12% DMSO (group B), Cell Banker 1 (group C), and Cell Banker 2 (group D). The hepatocytes in each group were thawed at 3 days, 10 days, and 5 months of cryopreservation and subjected to comparative analyses, including viability, plating efficiency, LDH release, ammonia removal test, and lentiviral gene transfer. These parameters were the most favorable in the hepatocytes cryopreserved with UW solution. Approximately 5% of thawed cryopreserved porcine hepatocytes expressed LacZ activity after lentiviral transduction. Intrasplenic transplantation of UW solution-cryopreserved hepatocytes improved the survival of rats treated with D-galactosamine. UW solution maintained the functions of cryopreserved porcine hepatocytes.

  12. RNAi in murine hepatocytes: the agony of choice--a study of the influence of lipid-based transfection reagents on hepatocyte metabolism.

    PubMed

    Böttger, Jan; Arnold, Katrin; Thiel, Carlo; Rennert, Christiane; Aleithe, Susanne; Hofmann, Ute; Vlaic, Sebastian; Sales, Susanne; Shevchenko, Andrej; Matz-Soja, Madlen

    2015-09-01

    Primary hepatocyte cell cultures are widely used for studying hepatic diseases with alterations in hepatic glucose and lipid metabolism, such as diabetes and non-alcoholic fatty liver disease. Therefore, small interfering RNAs (siRNAs) provide a potent and specific tool to elucidate the signaling pathways and gene functions involved in these pathologies. Although RNA interference (RNAi) in vitro is frequently used in these investigations, the metabolic alterations elucidated by different siRNA delivery strategies have hardly been investigated in transfected hepatocytes. To elucidate the influence of the most commonly used lipid-based transfection reagents on cultured primary hepatocytes, we studied the cytotoxic effects and transfection efficiencies of INTERFERin(®), Lipofectamine(®)RNAiMAX, and HiPerFect(®). All of these transfection agents displayed low cytotoxicity (5.6-9.0 ± 1.3-3.4%), normal cell viability, and high transfection efficiency (fold change 0.08-0.13 ± 0.03-0.05), and they also favored the satisfactory down-regulation of target gene expression. However, when effects on the metabolome and lipidome were studied, considerable differences were observed among the transfection reagents. Cellular triacylglycerides levels were either up- or down-regulated [maximum fold change: INTERFERin(®) (48 h) 2.55 ± 0.34, HiPerFect(®) (24 h) 0.79 ± 0.08, Lipofectamine(®)RNAiMAX (48 h) 1.48 ± 0.21], and mRNA levels of genes associated with lipid metabolism were differentially affected. Likewise, metabolic functions such as amino acid utilization from were perturbed (alanine, arginine, glycine, ornithine, and pyruvate). In conclusion, these findings demonstrate that the choice of non-viral siRNA delivery agent is critical in hepatocytes. This should be remembered, especially if RNA silencing is used for studying hepatic lipid homeostasis and its regulation.

  13. 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

  14. Characterization of the RokA and HexA broad-substrate-specificity hexokinases from Bacteroides fragilis and their role in hexose and N-acetylglucosamine utilization.

    PubMed

    Brigham, Christopher J; Malamy, Michael H

    2005-02-01

    Bacteroides fragilis, a human gastrointestinal commensal and an opportunistic pathogen, utilizes simple and complex sugars and polysaccharides for growth in the large intestine and at sites of infection. Because B. fragilis lacks transport-linked sugar phosphorylation systems, cytoplasmic kinase(s) was expected to be required for the phosphorylation of hexoses and hexosamines. We have now identified two hexose kinases that are important for growth of B. fragilis on glucose, mannose, and other sugars. One kinase (RokA), a member of the ROK family of proteins, was found to be the sole kinase for activation of N-acetyl-D-glucosamine (NAG). The other kinase (HexA) is responsible for the majority of the glucose kinase activity in the cell, although a hexA deletion mutant strain was not defective for growth on any substrate tested. Deletion of both the rokA and hexA kinase genes resulted in inability of the cell to use glucose, mannose, NAG, and many other sugars. We purified RokA and determined its approximate molecular mass to be 36.5 kDa. The purified RokA protein was shown to phosphorylate several substrates, including glucose, NAG, and mannose, but not N-acetylmannosamine or N-acetylneuraminic acid. Phylogenetic analysis of RokA showed that it is most similar to kinases from the Cytophaga-Flavibacterium-Bacteroides group, while HexA was most similar to other bacterial hexokinases and eukaryotic hexokinases.

  15. Autocrine expression of hepatocyte growth factor and its cytoprotective effect on hepatocyte poisoning

    PubMed Central

    He, Yong; Zhou, Jun; Dou, Ke-Feng; Chen, Yong; Yan, Qing-Guo; Li, Hai-Min

    2004-01-01

    AIM: To construct pEGFP-hepatocyte growth factor (HGF) expression vector, the to detect its expression in transfected human hepatocytes, and to investigate the influence of autocrine HGF expression on the proliferative potential and cytoprotective effects in human hepatocytes. METHODS: Human HGF cDNA was ligated to the pEGFP vector. Recombinant plasmid was transfected into human hepatocyte line QZG with liposome. Expression of HGF protein was observed by fluorescence microscopy and immunohistochemistry. Hepatic cells were collected 24, 48, and 72 h after transfection to detect the number of [3H]-TdR uptake in DNA. DNA synthesis was observed by using PCNA stain immunohistochemistry. Acute liver cell damage was induced by carbon tetrachloride. Cytoprotective effect was observed by examining the survival rate of hepatocytes and leakage of intracellular alanine transaminase (ALT) and potassium ions. RESULTS: HGF identification of pEGFP-HGF by enzyme digestion showed that HGF fragment was cloned into BamH I and Sal I sites of pEGFP-N3. Expression of GFP in transfected hepatocytes was observed with fluorescence microscopy. The [3H]-TdR uptake became 7 times as many as in the control group 96 h after transfection. After HGF transfection, the survival rate of hepatocytes poisoned by CCl4 significantly increased (83% vs 61%, P < 0.05), and the leakage of intracellular alanine transaminase and potassium ions decreased (586 nkat/L vs 1089 nkat/L, P < 0.01; and 5.59 mmol/L vs 6.02 mmol/L, P < 0.01 respectively). Culture of transfected hepatic cells promoted the proliferation of other non-transfected cells. CONCLUSION: Transfected HGF is expressed in hepatic cells and has the activity of promoting cell division and protecting hepatic cells against poisoning. PMID:15334679

  16. Hepatocyte Culture in Autologous Decellularized Spleen Matrix

    PubMed Central

    Gao, Rui; Wu, Wanquan; Xiang, Junxi; Lv, Yi; Zheng, Xinglong; Chen, Qian; Wang, Haohua; Wang, Bo; Liu, Zhengwen; Ma, Feng

    2015-01-01

    abstract Background and Aims: Using decellularized scaffold to reengineer liver tissue is a promising alternative therapy for end-stage liver diseases. Though the decellularized human liver matrix is the ideal scaffold for reconstruction of the liver theoretically, the shortage of liver donors is still an obstacle for potential clinical application. Therefore, an appropriate alternative scaffold is needed. In the present study, we used a tissue engineering approach to prepare a rat decellularized spleen matrix (DSM) and evaluate the effectiveness of this DSM for primary rat hepatocytes culture. Methods: Rat decellularized spleen matrix (DSM) was prepared by perfusion of a series of detergents through spleen vasculature. DSM was characterized by residual DNA and specific extracellular matrix distribution. Thereafter, primary rat hepatocytes were cultured in the DSM in a 3-dimensional dynamic culture system, and liver cell survival and biological functions were evaluated by comparison with 3-dimensional sandwich culture and also with cultured in decellularized liver matrix (DLM). Results: Our research found that DSM did not exhibit any cellular components, but preserved the main extracellular matrix and the intact vasculature evaluated by DNA detection, histology, immunohistochemical staining, vessel corrosion cast and upright metallurgical microscope. Moreover, the method of DSM preparation procedure was relatively simple with high success rate (100%). After seeding primary hepatocytes in DSM, the cultured hepatocytes survived inside DSM with albumin synthesis and urea secretion within 10 d. Additionally, hepatocytes in dynamic culture medium had better biological functions at day 10 than that in sandwich culture. Albumin synthesis was 85.67 ± 6.34 μg/107cell/24h in dynamic culture in DSM compared to 62.43 ± 4.59 μg/107cell/24h in sandwich culture (P < 0.01) and to 87.54 ± 5.25 μg/107cell/24h in DLM culture (P > 0.05); urea release was 32.14 ± 8.62

  17. Hepatocyte growth factor (HGF) and hemodialysis: physiopathology and clinical implications.

    PubMed

    Libetta, Carmelo; Esposito, Pasquale; Martinelli, Claudia; Grosjean, Fabrizio; Gregorini, Marilena; Rampino, Teresa; Dal Canton, Antonio

    2016-06-01

    Hepatocyte growth factor (HGF) is a pleiotropic cytokine which exerts a variety of effects on several cells, being involved in the regulation of many biological processes, such as inflammation, tissue repair, morphogenesis, angiogenesis, tumour propagation, immunomodulation of viral infections and cardio-metabolic activities. Patients undergoing regular hemodialysis (HD) present elevated levels of HGF, mainly due to the leukocyte activation associated with HD treatment. High HGF levels might account for specific clinical features of HD patients, i.e. mild liver damage in course of HCV-infection and high cardiovascular risk profile. Moreover, in patients with acute kidney injury, the induction of HGF may represent a crucial step to promote renal recovery, which can have important prognostic consequences in the short and long-term. In this review we discuss the mechanisms underlying HGF production in HD patients, the role of HGF in this particular patient population and the potential clinical implications derived from the study of HGF in HD patients.

  18. 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

  19. Prox1 ablation in hepatic progenitors causes defective hepatocyte specification and increases biliary cell commitment

    PubMed Central

    Seth, Asha; Ye, Jianming; Yu, Nanjia; Guez, Fanny; Bedford, David C.; Neale, Geoffrey A.; Cordi, Sabine; Brindle, Paul K.; Lemaigre, Frederic P.; Kaestner, Klaus H.; Sosa-Pineda, Beatriz

    2014-01-01

    The liver has multiple functions that preserve homeostasis. Liver diseases are debilitating, costly and often result in death. Elucidating the developmental mechanisms that establish the liver’s architecture or generate the cellular diversity of this organ should help advance the prevention, diagnosis and treatment of hepatic diseases. We previously reported that migration of early hepatic precursors away from the gut epithelium requires the activity of the homeobox gene Prox1. Here, we show that Prox1 is a novel regulator of cell differentiation and morphogenesis during hepatogenesis. Prox1 ablation in bipotent hepatoblasts dramatically reduced the expression of multiple hepatocyte genes and led to very defective hepatocyte morphogenesis. As a result, abnormal epithelial structures expressing hepatocyte and cholangiocyte markers or resembling ectopic bile ducts developed in the Prox1-deficient liver parenchyma. By contrast, excessive commitment of hepatoblasts into cholangiocytes, premature intrahepatic bile duct morphogenesis, and biliary hyperplasia occurred in periportal areas of Prox1-deficient livers. Together, these abnormalities indicate that Prox1 activity is necessary to correctly allocate cell fates in liver precursors. These results increase our understanding of differentiation anomalies in pathological conditions and will contribute to improving stem cell protocols in which differentiation is directed towards hepatocytes and cholangiocytes. PMID:24449835

  20. 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.

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

    PubMed Central

    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

  2. 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).

  3. Experimental hepatocyte xenotransplantation--a comprehensive review of the literature.

    PubMed

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

    2015-01-01

    Hepatocyte transplantation (Tx) 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 Tx. Hepatocytes from other species, for example, 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 xenoTx. The literature search identified 51 reports of in vivo cross-species Tx of hepatocytes in a variety of experimental models. Most studies investigated the Tx 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 Tx (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. The data provided by this literature search strengthen the hypothesis that xenoTx 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 xenoTx.

  4. Hepatocyte xenotransplantation for treating liver disease.

    PubMed

    Bonavita, André Gustavo; Quaresma, Kátia; Cotta-de-Almeida, Vinícius; Pinto, Marcelo Alves; Saraiva, Roberto Magalhães; Alves, Luiz Anastácio

    2010-01-01

    The treatment of acute and chronic liver failure is still a challenge despite modern therapeutic innovations. While liver transplantation can restore liver function and improve patient survival, donor shortages limit this treatment to a small number of patients. Cellular xenotransplantation has emerged as an alternative for treating liver failure. Xenohepatocytes could be readily available in sufficient quantities to treat patients in critical condition and thereby reduce the donor shortage. The use of isolated encapsulated or non-encapsulated cells can reduce the immunorejection response. Several studies using animal models of acute or chronic liver failure have demonstrated improved survival and recovery of liver function after xenotransplantation of adult hepatocytes. Porcine liver cells are a potential source of xenohepatocytes due to similarities with human physiology and the great number of hepatocytes that can be obtained. The recent development of less immunogenic transgenic pigs, new immunosuppressive drugs, and cellular encapsulation systems represents important advances in the field of cellular xenotransplantation. In this study, we review the work carried out in animal models that deals with the advantages and limitations of hepatocyte xenotransplantation, and we propose new studies needed in this field.

  5. Sustained endoplasmic reticulum stress inhibits hepatocyte proliferation via downregulation of c-Met expression.

    PubMed

    He, Yihuai; Long, Jun; Zhong, Weiwei; Fu, Yu; Li, Ying; Lin, Shide

    2014-04-01

    The molecular mechanisms of impaired liver regeneration in several liver diseases remain poorly understood. Endoplasmic reticulum (ER) stress has been observed in a variety of liver diseases. The aims of this study were to explore the impacts of ER stress on hepatocyte growth factor (HGF)-induced proliferation and c-Met expression in human hepatocyte L02 cells. Human hepatocyte L02 cells were incubated with thapsigargin (TG) to induce ER stress. 4-Phenylbutyric acid (PBA) was used to rescue ER stress. Activation of glucose-regulated protein 78, phosphorylation of PKR-like ER kinase and eukaryotic translation initiation factor-2α, and the expression of c-Met were determined by western blotting. The expression of c-Met mRNA was observed by reverse transcription polymerase chain reaction. L02 cell proliferation was determined by the MTS assay. L02 cell proliferation was significantly impaired in TG-treated L02 cells from 24 to 48 h, while PBA partly restored the proliferation of L02 cells. In addition, TG treatment significantly decreased the sensitivity of L02 cells to HGF-induced proliferation. PBA partly resumed the sensitivity of L02 cells to HGF-induced proliferation. The expression of c-Met protein in L02 cells was downregulated from 6 h after TG treatment, and PBA partly restored c-Met expression inhibited by TG. The expression of c-Met mRNA was also significantly downregulated from 24 to 48 h after TG treatment. Our results strongly suggest that sustained ER stress inhibits hepatocyte proliferation via downregulation of both c-Met mRNA and protein expression in human hepatocyte L02 cells.

  6. Engineered glucagon-like peptide-1-producing hepatocytes lower plasma glucose levels in mice.

    PubMed

    Riedel, Michael J; Lee, Corinna Wai Kwan; Kieffer, Timothy J

    2009-04-01

    Glucagon-like peptide (GLP)-1 is an incretin hormone with well-characterized antidiabetic properties, including glucose-dependent stimulation of insulin secretion and enhancement of beta-cell mass. GLP-1 agonists have recently been developed and are now in clinical use for the treatment of type 2 diabetes. Rapid degradation of GLP-1 by enzymes including dipeptidyl-peptidase (DPP)-IV and neutral endopeptidase (NEP) 24.11, along with renal clearance, contribute to a short biological half-life, necessitating frequent injections to maintain therapeutic efficacy. Gene therapy may represent a promising alternative approach for achieving long-term increases in endogenous release of GLP-1. We have developed a novel strategy for glucose-regulated production of GLP-1 in hepatocytes by expressing a DPP-IV-resistant GLP-1 peptide in hepatocytes under control of the liver-type pyruvate kinase promoter. Adenoviral delivery of this construct to hepatocytes in vitro resulted in production and secretion of bioactive GLP-1 as measured by a luciferase-based bioassay developed to detect the NH2-terminally modified GLP-1 peptide engineered for this study. Transplantation of encapsulated hepatocytes into CD-1 mice resulted in an increase in plasma GLP-1 levels that was accompanied by a significant reduction in fasting plasma glucose levels. The results from this study demonstrate that a gene therapy approach designed to induce GLP-1 production in hepatocytes may represent a novel strategy for long-term secretion of bioactive GLP-1 for the treatment of type 2 diabetes.

  7. Structural and functional hepatocyte polarity and liver disease

    PubMed Central

    Gissen, Paul; Arias, Irwin M.

    2015-01-01

    Summary Hepatocytes form a crucially important cell layer that separates sinusoidal blood from the canalicular bile. They have a uniquely organized polarity with a basal membrane facing liver sinusoidal endothelial cells, while one or more apical poles can contribute to several bile canaliculi jointly with the directly opposing hepatocytes. Establishment and maintenance of hepatocyte polarity is essential for many functions of hepatocytes and requires carefully orchestrated cooperation between cell adhesion molecules, cell junctions, cytoskeleton, extracellular matrix and intracellular trafficking machinery. The process of hepatocyte polarization requires energy and, if abnormal, may result in severe liver disease. A number of inherited disorders affecting tight junction and intracellular trafficking proteins have been described and demonstrate clinical and pathophysiological features overlapping those of the genetic cholestatic liver diseases caused by defects in canalicular ABC transporters. Thus both structural and functional components contribute to the final hepatocyte polarity phenotype. Many acquired liver diseases target factors that determine hepatocyte polarity, such as junctional proteins. Hepatocyte depolarization frequently occurs but is rarely recognized because hematoxylin-eosin staining does not identify the bile canaliculus. However, the molecular mechanisms underlying these defects are not well understood. Here we aim to provide an update on the key factors determining hepatocyte polarity and how it is affected in inherited and acquired diseases. PMID:26116792

  8. Hepatocyte transplantation program: Lessons learned and future strategies

    PubMed Central

    Ibars, Eugenia Pareja; Cortes, Miriam; Tolosa, Laia; Gómez-Lechón, Maria José; López, Slivia; Castell, José Vicente; Mir, José

    2016-01-01

    This review aims to share the lessons we learned over time during the setting of the hepatocyte transplantation (HT) program at the Hepatic Cell Therapy Unit at Hospital La Fe in Valencia. New sources of liver tissue for hepatocyte isolation have been explored. The hepatocyte isolation and cryopreservation procedures have been optimized and quality criteria for assessment of functionality of hepatocyte preparations and suitability for HT have been established. The results indicate that: (1) Only highly viable and functional hepatocytes allow to recover those functions lacking in the native liver; (2) Organs with steatosis (≥ 40%) and from elderly donors are declined since low hepatocyte yields, viability and cell survival after cryopreservation, are obtained; (3) Neonatal hepatocytes are cryopreserved without significant loss of viability or function representing high-quality cells to improve human HT; (4) Cryopreservation has the advantage of providing hepatocytes constantly available and of allowing the quality evaluation and suitability for transplantation; and (5) Our results from 5 adults with acute liver failure and 4 from children with inborn metabolic diseases, indicate that HT could be a very useful and safe cell therapy, as long as viable and metabolically functional human hepatocytes are used. PMID:26811633

  9. Separation and characterization of the aldehydic products of lipid peroxidation stimulated by carbon tetrachloride or ADP-iron in isolated rat hepatocytes and rat liver microsomal suspensions.

    PubMed Central

    Poli, G; Dianzani, M U; Cheeseman, K H; Slater, T F; Lang, J; Esterbauer, H

    1985-01-01

    Carbonyl products were separated and identified in suspensions of rat liver microsomal fractions and in isolated hepatocytes, after stimulation of lipid peroxidation by incubation with the pro-oxidants CCl4 and ADP-iron. The carbonyl products were allowed to react with 2,4-dinitrophenylhydrazine, and the derivatives were extracted and separated by t.l.c. into three zones of non-polar materials, and one fraction of polar derivatives that remained at the origin. Separation of the individual non-polar hydrazones in each zone by h.p.l.c. demonstrated that zone I prepared from microsomal fraction or hepatocytes incubated with CCl4 or ADP-iron contained mainly 4-hydroxyhex-2-enal, 4-hydroxynon-2-enal and 4-hydroxynona-2,5-dienal. Zone III consisted mainly of the alkanals propanal, pentanal and hexanal, the 2-alkenals propenal, pent-2-enal, hex-2-enal, hept-2-enal, oct-2-enal and non-2-enal, the ketones butanone, pentan-2-one and pentan-3-one, and deca-2,4-dienal. Incubation of a microsomal fraction with ADP-iron was much more effective in producing malonaldehyde and other carbonyl products than an incubation with CCl4. Despite such quantitative differences, there were no obvious qualitative differences in the h.p.l.c. spectra obtained from zones I and III. However, the stoichiometric evaluation of fatty acid loss and the production of malonaldehyde and other carbonyls suggests that the pathways of lipid peroxidation triggered by CCl4 and ADP-iron are different. The accumulation of carbonyl products of lipid peroxidation in isolated hepatocytes is strongly affected by their metabolism; in particular, 4-hydroxyalkenals were found to be metabolized very rapidly. Nonetheless, both CCl4 and ADP-iron produced stimulation in the production of malonaldehyde and non-polar carbonyl production. After incubation of rat hepatocytes with CCl4 or ADP-iron it was found that approx. 50% of the total amount of non-polar carbonyls produced during incubation escaped into the external medium

  10. Modulation of hepatocyte phenotype in vitro via chemomechanical tuning of polyelectrolyte multilayers

    PubMed Central

    Chen, Alice A.; Khetani, Salman R.; Lee, Sunyoung; Bhatia, Sangeeta N.; Van Vliet, Krystyn J.

    2009-01-01

    It is increasingly appreciated that since cell and tissue functions are regulated by chemomechanical stimuli, precise control over such stimuli will improve the functionality of tissue models. However, due to the inherent difficulty in decoupling these cues as presented by extracellular materials, few studies have explored the independent modulation of biochemical and mechanical stimuli towards the manipulation of sustained cellular processes. Here, we demonstrate that both mechanical compliance and ligand presentation of synthetic, weak polyelectrolyte multilayers (PEMs) can be tuned independently to influence the adhesion and liver-specific functions of primary rat hepatocytes over extended in vitro culture (two weeks). These synthetic PEMs exhibited elastic moduli E ranging over 200 kPa -< E < 142 MPa, as much as one thousand-fold more compliant than tissue-culture polystyrene (E ∼ 2.5 GPa). The most compliant of these PEM substrata promoted hepatocyte adhesion and spheroidal morphology. Subsequent modification of PEMs with type I collagen and the proteoglycan decorin did not alter substrata compliance, but enhanced the retention of spheroids on surfaces and stabilized hepatic functions (albumin and urea secretion, CYP450 detoxification activity). Decorin exhibited unique compliance-mediated effects on hepatic functions, down-regulating the hepatocyte phenotype when presented on highly compliant substrata while up-regulating hepatocyte functions when presented on increasingly stiffer substrata. These results show that phenotypic functions of liver models can be modulated by leveraging synthetic polymers to study and optimize the interplay of biochemical and mechanical cues at the cell–material interface. More broadly, these results suggest an enabling approach for the systematic design of functional tissue models applied to drug screening, cell-based therapies and fundamental studies in development, physiology and disease. PMID:19046762

  11. Effects of nutritional and hormonal factors on the metabolism of retinol-binding protein by primary cultures of rat hepatocytes

    SciTech Connect

    Dixon, J.L.; Goodman, D.S.

    1987-01-01

    Studies were conducted to explore hormonal and nutritional factors that might be involved in the regulation of retinol-binding protein (RBP) synthesis and secretion by the liver. The studies employed primary cultures of hepatocytes from normal rats. When cells were cultured in Dulbecco's modified Eagle's medium alone, a high rate of RBP secretion was observed initially, which declined and became quite low by 24 hr. Supplementing the medium with amino acids maintained RBP and albumin secretion at moderate (but less than initial) rates for at least 3 days. Further addition of dexamethasone maintained the production and secretion rates of RBP, transthyretin, and albumin close to the initial rates for up to 3-5 days in culture as measured by radioimmunoassay. Hormonally treated hepatocytes produced and secreted RBP, transthyretin, and albumin at both absolute and relative rates similar to physiological values, as estimated from rates reported by others from studies in vivo and with perfused livers. Glucagon addition partially maintained the secretion rates of these 3 proteins, but less effectively than did dexamethasone. A number of other hormones, added singly or in combination, did not affect RBP production or secretion. Addition of retinol to the cultured normal hepatocytes was without effect upon RBP secretion. These studies show that supplementing the culture medium of hepatocytes with amino acids and dexamethasone maintains RBP production and secretion for several days. In normal hepatocytes, with ample supply of retinol available within the cell, addition of exogenous retinol does not appear to influence RBP metabolism or secretion by the cells.

  12. Programmed hepatocytes cell death associated with FLIP downregulation in response to extracellular preS1/2.

    PubMed

    Rojas, Masyelly D; Peterson, Darrell L; Barboza, Luisa; Terán-Ángel, Guillermo; Labastida-Moreno, Cesar A; Berrueta, Lisbeth; Salmen, Siham

    2014-03-01

    Chronic hepatitis B virus (HBV) infection involves liver damage resulting in continuous cell injury and death. During HBV infection, hepatocytes exhibit changes in death receptor expression and in their susceptibility to death. These changes are observed not only in infected cells but also in bystander cells. Because excess viral surface protein (HBsAg) is secreted in large amounts as soluble particles containing preS proteins, the role of soluble preS1/2 in hepatocyte (HepG2) death modulation is an important issue to be explored. An increase of cell death induced by preS1/2 was observed. Also, cell death was associated with the down-regulation of FLIP and activation of caspase 8, caspase 9, and BID. Additionally, hepatocytes exhibited a sensitization to death mediated by the Fas receptor. These results, may contribute to understanding the role of envelope proteins (preS1/2) in the pathogenesis of HBV infection.

  13. [Encapsulating hepatocytes with chitosan in physiological conditions].

    PubMed

    Zhu, Jianhang; Zhang, Bao; Yan, Xiluan; Lao, Xuejun; Yu, Hanry

    2006-10-01

    Prepared from 15.3% N-acetylated chitosan (FNC), half N-acetylated chitosan (HNC) possesses a good solubility in a weak basic solution, guaranteeing the formation of microcapsules by the coacervating reaction between HNC and methacrylic acid (MAA)-hydroxyethyl methacrylate (HEMA)-methyl methacrylate (MMA) (MAA-HEMA-MMA) terpolymer under physiological conditions. When hepatocytes were encapsulated in such 3-dimensional microenvironment, as compared to monolayer culture, cell functions, including P450 activity, urea production and albumin release, were well supported. The prepared microcapsules have good mechanical stability and permeability.

  14. Oxidative Stress in the Healthy and Wounded Hepatocyte: A Cellular Organelles Perspective

    PubMed Central

    Mello, Tommaso; Zanieri, Francesca; Ceni, Elisabetta; Galli, Andrea

    2016-01-01

    Accurate control of the cell redox state is mandatory for maintaining the structural integrity and physiological functions. This control is achieved both by a fine-tuned balance between prooxidant and anti-oxidant molecules and by spatial and temporal confinement of the oxidative species. The diverse cellular compartments each, although structurally and functionally related, actively maintain their own redox balance, which is necessary to fulfill specialized tasks. Many fundamental cellular processes such as insulin signaling, cell proliferation and differentiation and cell migration and adhesion, rely on localized changes in the redox state of signal transducers, which is mainly mediated by hydrogen peroxide (H2O2). Therefore, oxidative stress can also occur long before direct structural damage to cellular components, by disruption of the redox circuits that regulate the cellular organelles homeostasis. The hepatocyte is a systemic hub integrating the whole body metabolic demand, iron homeostasis and detoxification processes, all of which are redox-regulated processes. Imbalance of the hepatocyte's organelles redox homeostasis underlies virtually any liver disease and is a field of intense research activity. This review recapitulates the evolving concept of oxidative stress in the diverse cellular compartments, highlighting the principle mechanisms of oxidative stress occurring in the healthy and wounded hepatocyte. PMID:26788252

  15. A Hemoglobin Based Oxygen Carrier, Bovine Polymerized Hemoglobin (HBOC-201) versus Hetastarch (HEX) in an Uncontrolled Liver Injury Hemorrhagic Shock Swine Model with Delayed Evacuation

    DTIC Science & Technology

    2004-10-01

    A Hemoglobin Based Oxygen Carrier, Bovine Polymerized Hemoglobin (HBOC-201) versus Hetastarch (HEX) in an Uncontrolled Liver Injury Hemorrhagic Shock...Transcutaneous tis- sue oxygenation was restored more rap- idly in HBOC-201 pigs, there was a trend to lower lactic acid, and base deficit was less...lactic acidosis and base deficit (BD) abnormalities, indicating on-going hypoperfusion.2–4 As these abnormalities measured upon hospital arrival

  16. Identification of the medicinal off-flavor compound formed from ascorbic acid and (E)-hex-2-enal.

    PubMed

    Sakamaki, Kensuke; Ishizaki, Susumu; Ohkubo, Yasutaka; Tateno, Yoshiyuki; Fujita, Akira

    2011-06-22

    A test apple beverage made up of apple juice (20%), high-fructose corn syrup (11.5%), citric acid (0.43%), trisodium citrate (0.02%), apple-odor flavor (0.1%), and ascorbic acid (0.02%) was stored at 40 °C and then analyzed for the change of odor in the beverage. Although no thermoacidophilic bacteria (TAB) were detected, a medicinal off-flavor was perceived after the 8 weeks of storage. Model experiments on the ingredients of the test apple beverage revealed that the off-flavor compound had been formed by ascorbic acid and (E)-hex-2-enal. Synthesis and NMR (¹H, ¹³C, HMQC, and HMBC) analyses identified the compound as 6-propylbenzofuran-7-ol. The odor quality, retention index (RI), and mass spectrum of synthetic 6-propylbenzofuran-7-ol were identical with those of the medicinal odor compound from the test apple beverage. Sensory evaluation revealed the recognition thresholds for medicinal odor were 31.4 ppb in water and 24.0 ppb in apple beverage, and the detection thresholds were 19.6 ppb in water and 8.6 ppb in apple beverage, respectively. The quantified concentration of 6-propylbenzofuran-7-ol formed in test apple beverage was 90 ppb, approximately. This concentration was well above the odor threshold, so it was concluded that the compound was the source of the medicinal off-flavor.

  17. Hepatocyte nuclear factor 4alpha orchestrates expression of cell adhesion proteins during the epithelial transformation of the developing liver.

    PubMed

    Battle, Michele A; Konopka, Genevieve; Parviz, Fereshteh; Gaggl, Alexandra Lerch; Yang, Chuhu; Sladek, Frances M; Duncan, Stephen A

    2006-05-30

    Epithelial formation is a central facet of organogenesis that relies on intercellular junction assembly to create functionally distinct apical and basal cell surfaces. How this process is regulated during embryonic development remains obscure. Previous studies using conditional knockout mice have shown that loss of hepatocyte nuclear factor 4alpha (HNF4alpha) blocks the epithelial transformation of the fetal liver, suggesting that HNF4alpha is a central regulator of epithelial morphogenesis. Although HNF4alpha-null hepatocytes do not express E-cadherin (also called CDH1), we show here that E-cadherin is dispensable for liver development, implying that HNF4alpha regulates additional aspects of epithelial formation. Microarray and molecular analyses reveal that HNF4alpha regulates the developmental expression of a myriad of proteins required for cell junction assembly and adhesion. Our findings define a fundamental mechanism through which generation of tissue epithelia during development is coordinated with the onset of organ function.

  18. A Review of X-ray Diagnostic Calibrations in the 2 to 100 keV Region Using the High Energy X-ray Calibration Facility (HEX)

    SciTech Connect

    Ali, Zaheer; Pond, T; Buckles, R A; Maddox, B R; Chen, C D; DeWald, E L; Izumi, N; Stewart, R

    2010-05-19

    The precise and accurate measurement of X-rays in the 2 keV to 100 keV region is crucial to the understanding of HED plasmas and warm dense matter in general. With the emergence of inertially confined plasma facilities as the premier platforms for ICF, laboratory astrophysics, and national security related plasma experiments, the need to calibrate diagnostics in the high energy X-ray regime has grown. At National Security Technologies High Energy X-ray Calibration Facility (HEX) in Livermore, California, X-ray imagers, filter-fluorescer spectrometers, crystal spectrometers, image plates, and nuclear diagnostics are calibrated. The HEX can provide measurements of atomic line radiation, X-ray flux (accuracy within 10%), and X-ray energy (accuracy within 1%). The HEX source is comprised of a commercial 160 kV X-ray tube, a fluorescer wheel, a filter wheel, and a lead encasement. The X-ray tube produces a Tungsten bremsstrahlung spectrum which causes a foil to fluoresce line radiation. To minimize bremsstrahlung in the radiation for calibration we also provide various foils as filters. For experimental purposes, a vacuum box capable of 10{sup -7} Torr, as well as HPGe and CdTe radiation detectors, are provided on an optical table. Most geometries and arrangements can be changed to meet experimental needs.

  19. Analysis of DNA methylation change induced by Dnmt3b in mouse hepatocytes.

    PubMed

    Takahashi, Mayumi; Kamei, Yasutomi; Ehara, Tatsuya; Yuan, Xunmei; Suganami, Takayoshi; Takai-Igarashi, Takako; Hatada, Izuho; Ogawa, Yoshihiro

    2013-05-17

    DNA methylation is a key epigenetic contributor to gene regulation in mammals. We have recently found that in the mouse liver, the promoter region of glycerol-3-phosphate acyltransferase 1, a rate-limiting enzyme of de novo lipogenesis, is regulated by DNA methylation, which is mediated by Dnmt3b, an enzyme required for the initiation of de novo methylation. In this study, using primary cultures of mouse hepatocytes with adenoviral overexpression of Dnmt3b, we characterized Dnmt3b-dependent DNA methylation on a genome-wide basis. A genome-wide DNA methylation analysis, called microarray-based integrated analysis of methylation by isoschizomers, identified 108 genes with Dnmt3b dependent DNA methylation. In DNA expression array analysis, expression of some genes with Dnmt3b-dependent DNA methylation was suppressed. Studies with primary mouse hepatocytes overexpressing Dnmt3b or Dnmt3a revealed that many genes with Dnmt3b-dependent methylation are not methylated by Dnmt3a, whereas those methylated by Dnmt3a are mostly methylated by Dnmt3b. Bioinformatic analysis showed that the CANAGCTG and CCGGWNCSC (N denotes A, T, G, or C; W denotes A or T; and S denotes C or G) sequences are enriched in genes methylated by overexpression of Dnmt3b and Dnmt3a, respectively. We also observed a large number of genes with Dnmt3b-dependent DNA methylation in primary cultures of mouse hepatocytes with adenoviral overexpression of Dnmt3, suggesting that Dnmt3b is an important DNA methyltransferase in primary mouse hepatocytes, targets specific genes, and potentially plays a role in vivo.

  20. Insulin internalization in isolated rat hepatocytes

    SciTech Connect

    Galan, J.; Trankina, M.; Noel, R.; Ward, W. )

    1990-02-26

    This project was designed to determine whether neomycin, an aminoglycoside antibiotic, has a significant effect upon the pathways of ligand endocytosis in isolated rat hepatocytes. The pathways studied include receptor-mediated endocytosis and fluid-phase endocytosis. Neomycin causes a dose-dependent acceleration of {sup 125}I-insulin internalization. Since fluid-phase endocytosis can also be a significant factor in {sup 125}I-insulin internalization, lucifer yellow (LY), a marker for fluid-phase endocytosis, was incorporated into an assay similar to the {sup 125}I-insulin internalization procedure. In the presence of 5 mM neomycin, a significant increase in LY uptake was evident at 0.2 and 0.4 mg/ml of LY. At 0.8 mg/ml, a decrease in LY uptake was observed. The increased rate of {sup 125}I-insulin internalization in the presence of neomycin was intriguing. Since one action of neomycin is to inhibit phosphoinositidase C, it suggests that the phosphotidylinositol cycle may be involved in ligand internalization by hepatocytes. At low insulin concentrations, receptor-mediated uptake predominates. Fluid-phase uptake can become an important uptake route as insulin concentrations are increased. Since neomycin stimulates fluid-phase endocytosis, it must also be taken into account when measuring ligand internalization.

  1. Mechanisms of the statins cytotoxicity in freshly isolated rat hepatocytes.

    PubMed

    Abdoli, Narges; Heidari, Reza; Azarmi, Yadollah; Eghbal, Mohammad Ali

    2013-06-01

    Statins are potent drugs, used as lipid-lowering agents in cardiovascular diseases. Hepatotoxicity is one of the serious adverse effects of statins, and the exact mechanism of hepatotoxicity is not yet clear. In this study, the cytotoxic effects of the most commonly used statins, that is, atorvastatin, lovastatin, and simvastatin toward isolated rat hepatocytes, were evaluated. Markers, such as cell death, reactive oxygen species (ROS) formation, lipid peroxidation, mitochondrial membrane potential, and the amount of reduced and oxidized glutathione in the statin-treated hepatocytes, were investigated. It was found that the statins caused cytotoxicity toward rat hepatocytes dose dependently. An elevation in ROS formation, accompanied by a significant amount of lipid peroxidation and mitochondrial depolarization, was observed. Cellular glutathione reservoirs were decreased, and a significant amount of oxidized glutathione was formed. This study suggests that the adverse effect of statins toward hepatocytes is mediated through oxidative stress and the hepatocytes mitochondria play an important role in the statin-induced toxicity.

  2. Specific binding of Fusarium graminearum Hex1 protein to untranslated regions of the genomic RNA of Fusarium graminearum virus 1 correlates with increased accumulation of both strands of viral RNA.

    PubMed

    Son, Moonil; Choi, Hoseong; Kim, Kook-Hyung

    2016-02-01

    The HEX1 gene of Fusarium graminearum was previously reported to be required for the efficient accumulation of Fusarium graminearum virus 1 (FgV1) RNA in its host. To investigate the molecular mechanism underlying the production of FgHEX1 and the replication of FgV1 viral RNA, we conducted electrophoretic mobility shift assays (EMSA) with recombinant FgHex1 protein and RNA sequences derived from various regions of FgV1 genomic RNA. These analyses demonstrated that FgHex1 and both the 5'- and 3'-untranslated regions of plus-strand FgV1 RNA formed complexes. To determine whether FgHex1 protein affects FgV1 replication, we quantified accumulation viral RNAs in protoplasts and showed that both (+)- and (-)-strands of FgV1 RNAs were increased in the over-expression mutant and decreased in the deletion mutant. These results indicate that the FgHex1 functions in the synthesis of both strands of FgV1 RNA and therefore in FgV1 replication probably by specifically binding to the FgV1 genomic RNA.

  3. Roles of hepatocyte nuclear factors in hepatitis B virus infection

    PubMed Central

    Kim, Doo Hyun; Kang, Hong Seok; Kim, Kyun-Hwan

    2016-01-01

    Approximately 350 million people are estimated to be persistently infected with hepatitis B virus (HBV) worldwide. HBV maintains persistent infection by employing covalently closed circular DNA (cccDNA), a template for all HBV RNAs. Chronic hepatitis B (CHB) patients are currently treated with nucleos(t)ide analogs such as lamivudine, adefovir, entecavir, and tenofovir. However, these treatments rarely cure CHB because they are unable to inhibit cccDNA transcription and inhibit only a late stage in the HBV life cycle (the reverse transcription step in the nucleocapsid). Therefore, an understanding of the factors regulating cccDNA transcription is required to stop this process. Among numerous factors, hepatocyte nuclear factors (HNFs) play the most important roles in cccDNA transcription, especially in the generation of viral genomic RNA, a template for HBV replication. Therefore, proper control of HNF function could lead to the inhibition of HBV replication. In this review, we summarize and discuss the current understanding of the roles of HNFs in the HBV life cycle and the upstream factors that regulate HNFs. This knowledge will enable the identification of new therapeutic targets to cure CHB. PMID:27610013

  4. Nuclear lactate dehydrogenase modulates histone modification in human hepatocytes

    SciTech Connect

    Castonguay, Zachary; Auger, Christopher; Thomas, Sean C.; Chahma, M’hamed; Appanna, Vasu D.

    2014-11-07

    Highlights: • Nuclear LDH is up-regulated under oxidative stress. • SIRT1 is co-immunoprecipitated bound to nuclear LDH. • Nuclear LDH is involved in histone deacetylation and epigenetics. - Abstract: It is becoming increasingly apparent that the nucleus harbors metabolic enzymes that affect genetic transforming events. Here, we describe a nuclear isoform of lactate dehydrogenase (nLDH) and its ability to orchestrate histone deacetylation by controlling the availability of nicotinamide adenine dinucleotide (NAD{sup +}), a key ingredient of the sirtuin-1 (SIRT1) deacetylase system. There was an increase in the expression of nLDH concomitant with the presence of hydrogen peroxide (H{sub 2}O{sub 2}) in the culture medium. Under oxidative stress, the NAD{sup +} generated by nLDH resulted in the enhanced deacetylation of histones compared to the control hepatocytes despite no discernable change in the levels of SIRT1. There appeared to be an intimate association between nLDH and SIRT1 as these two enzymes co-immunoprecipitated. The ability of nLDH to regulate epigenetic modifications by manipulating NAD{sup +} reveals an intricate link between metabolism and the processing of genetic information.

  5. Structural basis for agonism and antagonism of hepatocyte growth factor

    SciTech Connect

    Tolbert, W. David; Daugherty-Holtrop, Jennifer; Gherardi, Ermanno; Vande Woude, George; Xu, H. Eric

    2010-11-01

    Hepatocyte growth factor (HGF) is an activating ligand of the Met receptor tyrosine kinase, whose activity is essential for normal tissue development and organ regeneration but abnormal activation of Met has been implicated in growth, invasion, and metastasis of many types of solid tumors. HGF has two natural splice variants, NK1 and NK2, which contain the N-terminal domain (N) and the first kringle (K1) or the first two kringle domains of HGF. NK1, which is a Met agonist, forms a head-to-tail dimer complex in crystal structures and mutations in the NK1 dimer interface convert NK1 to a Met antagonist. In contrast, NK2 is a Met antagonist, capable of inhibiting HGF's activity in cell proliferation without clear mechanism. Here we report the crystal structure of NK2, which forms a 'closed' monomeric conformation through interdomain interactions between the N- domain and the second kringle domain (K2). Mutations that were designed to open up the NK2 closed conformation by disrupting the N/K2 interface convert NK2 from a Met antagonist to an agonist. Remarkably, this mutated NK2 agonist can be converted back to an antagonist by a mutation that disrupts the NK1/NK1 dimer interface. These results reveal the molecular determinants that regulate the agonist/antagonist properties of HGF NK2 and provide critical insights into the dimerization mechanism that regulates the Met receptor activation by HGF.

  6. Hepatocytes as in vitro test system to investigate metabolite patterns of pesticides in farmed rainbow trout and common carp: Comparison between in vivo and in vitro and across species.

    PubMed

    Bischof, Ina; Köster, Jessica; Segner, Helmut; Schlechtriem, Christian

    2016-09-01

    In vitro tools using isolated primary fish hepatocytes have been proposed as a useful model to study the hepatic metabolism of xenobiotics in fish. In order to evaluate the potential of in vitro fish hepatocyte assays to provide information on in vivo metabolite patterns of pesticides in farmed fish, the present study addressed the following questions: Are in vitro and in vivo metabolite patterns comparable? Are species specific differences of metabolite patterns in vivo reflected in vitro? Are metabolite patterns obtained from cryopreserved hepatocytes comparable to those from freshly isolated cells? Rainbow trout and common carp were dosed orally with feed containing the pesticide methoxychlor (MXC) for 14days. In parallel, in vitro incubations using suspensions of freshly isolated or cryopreserved primary hepatocytes obtained from both species were performed. In vivo and in vitro samples were analyzed by thin-layer chromatography with authentic standards supported by HPLC-MS. Comparable metabolite patterns from a qualitative perspective were observed in liver in vivo and in hepatocyte suspensions in vitro. Species specific differences of MXC metabolite patterns observed between rainbow trout and common carp in vivo were well reflected by experiments with hepatocytes in vitro. Finally, cryopreserved hepatocytes produced comparable metabolite patterns to freshly isolated cells. The results of this study indicate that the in vitro hepatocyte assay could be used to identify metabolite patterns of pesticides in farmed fish and could thus serve as a valuable tool to support in vivo studies as required for pesticides approval according to the EU regulation 1107.

  7. Hepatocyte growth factor/scatter factor effects on epithelia. Regulation of intercellular junctions in transformed and nontransformed cell lines, basolateral polarization of c-met receptor in transformed and natural intestinal epithelia, and induction of rapid wound repair in a transformed model epithelium.

    PubMed Central

    Nusrat, A; Parkos, C A; Bacarra, A E; Godowski, P J; Delp-Archer, C; Rosen, E M; Madara, J L

    1994-01-01

    Intestinal epithelial cells rest on a fibroblast sheath. Thus, factors produced by these fibroblasts may influence epithelial function in a paracrine fashion. We examined modulation of intestinal epithelial function by one such fibroblast product, scatter factor/hepatocyte growth factor (HGF/SF). This effect was studied in vitro by using model T84 intestinal epithelial cells. When applied to confluent T84 monolayers, HGF/SF attenuates transepithelial resistance to passive ion flow in a dose-dependent manner (maximum fall at 300 ng/ml, 28% control monolayer resistance, P < 0.001, ED50 of 1.2 nM), t1/2 of 20 h. This functional effect of HGF/SF and distribution of its receptor, c-met, are polarized to the basolateral membranes of T84 intestinal epithelial cells. HGF/SF effects on resistance are not attributable to altered transcellular resistance (opening of Cl- and/or basolateral K+ channels), cytotoxicity, or enhanced cell proliferation; they therefore represent specific regulation of paracellular tight junction resistance. Analysis with biochemically purified rodent HGF/SF and Madin-Darby canine kidney cells reveals that effects on paracellular tight junctions also occur in other nontransformed epithelia. Binding of HGF/SF to its receptor in T84 intestinal epithelial cells is accompanied by tyrosine phosphorylation of the receptor. Because loosening of intercellular junctions between cells could facilitate separation, spreading, and migration of epithelial cells during physiologic processes such as wound resealing, we determined the effects of HGF/SF on intestinal epithelial wound resealing using our previously published in vitro model (Nusrat, A., C. Delp, and J. L. Madara. 1992. J. Clin. Invest. 89:1501-1511). HGF/SF markedly enhanced wound closure (> 450% increase in rate, P < 0.001) by influencing the migratory and spreading response in not only cells adjoining the wound but also cells many positions removed from the wound. We thus speculate that HGF/SF may

  8. Exosome-Mediated Intercellular Communication between Hepatitis C Virus-Infected Hepatocytes and Hepatic Stellate Cells.

    PubMed

    Devhare, Pradip B; Sasaki, Reina; Shrivastava, Shubham; Di Bisceglie, Adrian M; Ray, Ranjit; Ray, Ratna B

    2017-03-15

    Fibrogenic pathways in the liver are principally regulated by activation of hepatic stellate cells (HSC). Fibrosis is associated with chronic hepatitis C virus (HCV) infection, although the mechanism is poorly understood. HSC comprise the major population of nonparenchymal cells in the liver. Since HCV does not replicate in HSC, we hypothesized that exosomes secreted from HCV-infected hepatocytes activate HSC. Primary or immortalized human hepatic stellate (LX2) cells were exposed to exosomes derived from HCV-infected hepatocytes (HCV-exo), and the expression of fibrosis-related genes was examined. Our results demonstrated that HCV-exo internalized to HSC and increased the expression of profibrotic markers. Further analysis suggested that HCV-exo carry miR-19a and target SOCS3 in HSC, which in turn activates the STAT3-mediated transforming growth factor β (TGF-β) signaling pathway and enhances fibrosis marker genes. The higher expression of miR-19a in exosomes was also observed from HCV-infected hepatocytes and in sera of chronic HCV patients with fibrosis compared to healthy volunteers and non-HCV-related liver disease patients with fibrosis. Together, our results demonstrated that miR-19a carried through the exosomes from HCV-infected hepatocytes activates HSC by modulating the SOCS-STAT3 axis. Our results implicated a novel mechanism of exosome-mediated intercellular communication in the activation of HSC for liver fibrosis in HCV infection.IMPORTANCE HCV-associated liver fibrosis is a critical step for end-stage liver disease progression. However, the molecular mechanisms for hepatic stellate-cell activation by HCV-infected hepatocytes are underexplored. Here, we provide a role for miR-19a carried through the exosomes in intercellular communication between HCV-infected hepatocytes and HSC in fibrogenic activation. Furthermore, we demonstrate the role of exosomal miR-19a in activation of the STAT3-TGF-β pathway in HSC. This study contributes to the

  9. Acrolein cytotoxicity in hepatocytes involves endoplasmic reticulum stress, mitochondrial dysfunction and oxidative stress

    SciTech Connect

    Mohammad, Mohammad K.; Avila, Diana; Zhang, Jingwen; Barve, Shirish; Arteel, Gavin; McClain, Craig; Joshi-Barve, Swati

    2012-11-15

    Acrolein is a common environmental, food and water pollutant and a major component of cigarette smoke. Also, it is produced endogenously via lipid peroxidation and cellular metabolism of certain amino acids and drugs. Acrolein is cytotoxic to many cell types including hepatocytes; however the mechanisms are not fully understood. We examined the molecular mechanisms underlying acrolein hepatotoxicity in primary human hepatocytes and hepatoma cells. Acrolein, at pathophysiological concentrations, caused a dose-dependent loss of viability of hepatocytes. The death was apoptotic at moderate and necrotic at high concentrations of acrolein. Acrolein exposure rapidly and dramatically decreased intracellular glutathione and overall antioxidant capacity, and activated the stress-signaling MAP-kinases JNK, p42/44 and p38. Our data demonstrate for the first time in human hepatocytes, that acrolein triggered endoplasmic reticulum (ER) stress and activated eIF2α, ATF-3 and -4, and Gadd153/CHOP, resulting in cell death. Notably, the protective/adaptive component of ER stress was not activated, and acrolein failed to up-regulate the protective ER-chaperones, GRP78 and GRP94. Additionally, exposure to acrolein disrupted mitochondrial integrity/function, and led to the release of pro-apoptotic proteins and ATP depletion. Acrolein-induced cell death was attenuated by N-acetyl cysteine, phenyl-butyric acid, and caspase and JNK inhibitors. Our data demonstrate that exposure to acrolein induces a variety of stress responses in hepatocytes, including GSH depletion, oxidative stress, mitochondrial dysfunction and ER stress (without ER-protective responses) which together contribute to acrolein toxicity. Our study defines basic mechanisms underlying liver injury caused by reactive aldehyde pollutants such as acrolein. -- Highlights: ► Human primary hepatocytes and cultured cell lines are used. ► Multiple cell death signaling pathways are activated by acrolein. ► Novel finding of

  10. Intracellular cholesterol-binding proteins enhance HDL-mediated cholesterol uptake in cultured primary mouse hepatocytes

    PubMed Central

    Storey, Stephen M.; McIntosh, Avery L.; Huang, Huan; Landrock, Kerstin K.; Martin, Gregory G.; Landrock, Danilo; Payne, H. Ross; Atshaves, Barbara P.; Kier, Ann B.

    2012-01-01

    A major gap in our knowledge of rapid hepatic HDL cholesterol clearance is the role of key intracellular factors that influence this process. Although the reverse cholesterol transport pathway targets HDL to the liver for net elimination of free cholesterol from the body, molecular details governing cholesterol uptake into hepatocytes are not completely understood. Therefore, the effects of sterol carrier protein (SCP)-2 and liver fatty acid-binding protein (L-FABP), high-affinity cholesterol-binding proteins present in hepatocyte cytosol, on HDL-mediated free cholesterol uptake were examined using gene-targeted mouse models, cultured primary hepatocytes, and 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol). While SCP-2 overexpression enhanced NBD-cholesterol uptake, counterintuitively, SCP-2/SCP-x gene ablation also 1) enhanced the rapid molecular phase of free sterol uptake detectable in <1 min and initial rate and maximal uptake of HDL free cholesterol and 2) differentially enhanced free cholesterol uptake mediated by the HDL3, rather than the HDL2, subfraction. The increased HDL free cholesterol uptake was not due to increased expression or distribution of the HDL receptor [scavenger receptor B1 (SRB1)], proteins regulating SRB1 [postsynaptic density protein (PSD-95)/Drosophila disk large tumor suppressor (dlg)/tight junction protein (ZO1) and 17-kDa membrane-associated protein], or other intracellular cholesterol trafficking proteins (steroidogenic acute response protein D, Niemann Pick C, and oxysterol-binding protein-related proteins). However, expression of L-FABP, the single most prevalent hepatic cytosolic protein that binds cholesterol, was upregulated twofold in SCP-2/SCP-x null hepatocytes. Double-immunogold electron microscopy detected L-FABP sufficiently close to SRB1 for direct interaction, similar to SCP-2. These data suggest a role for L-FABP in HDL cholesterol uptake, a finding confirmed with SCP-2

  11. Intracellular cholesterol-binding proteins enhance HDL-mediated cholesterol uptake in cultured primary mouse hepatocytes.

    PubMed

    Storey, Stephen M; McIntosh, Avery L; Huang, Huan; Landrock, Kerstin K; Martin, Gregory G; Landrock, Danilo; Payne, H Ross; Atshaves, Barbara P; Kier, Ann B; Schroeder, Friedhelm

    2012-04-15

    A major gap in our knowledge of rapid hepatic HDL cholesterol clearance is the role of key intracellular factors that influence this process. Although the reverse cholesterol transport pathway targets HDL to the liver for net elimination of free cholesterol from the body, molecular details governing cholesterol uptake into hepatocytes are not completely understood. Therefore, the effects of sterol carrier protein (SCP)-2 and liver fatty acid-binding protein (L-FABP), high-affinity cholesterol-binding proteins present in hepatocyte cytosol, on HDL-mediated free cholesterol uptake were examined using gene-targeted mouse models, cultured primary hepatocytes, and 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol). While SCP-2 overexpression enhanced NBD-cholesterol uptake, counterintuitively, SCP-2/SCP-x gene ablation also 1) enhanced the rapid molecular phase of free sterol uptake detectable in <1 min and initial rate and maximal uptake of HDL free cholesterol and 2) differentially enhanced free cholesterol uptake mediated by the HDL3, rather than the HDL2, subfraction. The increased HDL free cholesterol uptake was not due to increased expression or distribution of the HDL receptor [scavenger receptor B1 (SRB1)], proteins regulating SRB1 [postsynaptic density protein (PSD-95)/Drosophila disk large tumor suppressor (dlg)/tight junction protein (ZO1) and 17-kDa membrane-associated protein], or other intracellular cholesterol trafficking proteins (steroidogenic acute response protein D, Niemann Pick C, and oxysterol-binding protein-related proteins). However, expression of L-FABP, the single most prevalent hepatic cytosolic protein that binds cholesterol, was upregulated twofold in SCP-2/SCP-x null hepatocytes. Double-immunogold electron microscopy detected L-FABP sufficiently close to SRB1 for direct interaction, similar to SCP-2. These data suggest a role for L-FABP in HDL cholesterol uptake, a finding confirmed with SCP-2

  12. HIV-1 Nef Is Transferred from Expressing T Cells to Hepatocytic Cells through Conduits and Enhances HCV Replication

    PubMed Central

    Park, In-Woo; Fan, Yan; Luo, Xiaoyu; Ryou, Myoung-Gwi; Liu, Jinfeng; Green, Linden; He, Johnny J.

    2014-01-01

    HIV-1 infection enhances HCV replication and as a consequence accelerates HCV-mediated hepatocellular carcinoma (HCC). However, the precise molecular mechanism by which this takes place is currently unknown. Our data showed that infectious HIV-1 failed to replicate in human hepatocytic cell lines. No discernible virus replication was observed, even when the cell lines transfected with HIV-1 proviral DNA were co-cultured with Jurkat T cells, indicating that the problem of liver deterioration in the co-infected patient is not due to the replication of HIV-1 in the hepatocytes of the HCV infected host. Instead, HIV-1 Nef protein was transferred from nef-expressing T cells to hepatocytic cells through conduits, wherein up to 16% (average 10%) of the cells harbored the transferred Nef, when the hepatocytic cells were co-cultured with nef-expressing Jurkat cells for 24 h. Further, Nef altered the size and numbers of lipid droplets (LD), and consistently up-regulated HCV replication by 1.5∼2.5 fold in the target subgenomic replicon cells, which is remarkable in relation to the initially indolent viral replication. Nef also dramatically augmented reactive oxygen species (ROS) production and enhanced ethanol-mediated up-regulation of HCV replication so as to accelerate HCC. Taken together, these data indicate that HIV-1 Nef is a critical element in accelerating progression of liver pathogenesis via enhancing HCV replication and coordinating modulation of key intra- and extra-cellular molecules for liver decay. PMID:24911518

  13. Inhibition of Drp1 protects against senecionine-induced mitochondria-mediated apoptosis in primary hepatocytes and in mice.

    PubMed

    Yang, Xiao; Wang, Hua; Ni, Hong-Min; Xiong, Aizhen; Wang, Zhengtao; Sesaki, Hiromi; Ding, Wen-Xing; Yang, Li

    2017-03-02

    Pyrrolizidine alkaloids (PAs) are a group of compounds found in various plants and some of them are widely consumed in the world as herbal medicines and food supplements. PAs are potent hepatotoxins that cause irreversible liver injury in animals and humans. However, the mechanisms by which PAs induce liver injury are not clear. In the present study, we determined the hepatotoxicity and molecular mechanisms of senecionine, one of the most common toxic PAs, in primary cultured mouse and human hepatocytes as well as in mice. We found that senecionine administration increased serum alanine aminotransferase levels in mice. H&E and TUNEL staining of liver tissues revealed increased hemorrhage and hepatocyte apoptosis in liver zone 2 areas. Mechanistically, senecionine induced loss of mitochondrial membrane potential, release of mitochondrial cytochrome c as well as mitochondrial JNK translocation and activation prior to the increased DNA fragmentation and caspase-3 activation in primary cultured mouse and human hepatocytes. SP600125, a specific JNK inhibitor, and ZVAD-fmk, a general caspase inhibitor, alleviated senecionine-induced apoptosis in primary hepatocytes. Interestingly, senecionine also caused marked mitochondria fragmentation in hepatocytes. Pharmacological inhibition of dynamin-related protein1 (Drp1), a protein that is critical to regulate mitochondrial fission, blocked senecionine-induced mitochondrial fragmentation and mitochondrial release of cytochrome c and apoptosis. More importantly, hepatocyte-specific Drp1 knockout mice were resistant to senecionine-induced liver injury due to decreased mitochondrial damage and apoptosis. In conclusion, our results uncovered a novel mechanism of Drp1-mediated mitochondrial fragmentation in senecionine-induced liver injury. Targeting Drp1-mediated mitochondrial fragmentation and apoptosis may be a potential avenue to prevent and treat hepatotoxicity induced by PAs.

  14. EGCG attenuates pro-inflammatory cytokines and chemokines production in LPS-stimulated L02 hepatocyte.

    PubMed

    Liu, Qiaoli; Qian, Yun; Chen, Feng; Chen, Xiaoming; Chen, Zhi; Zheng, Min

    2014-01-01

    Endotoxin lipopolysaccharide (LPS) plays an important role in the acceleration of inflammatory reaction of hepatitis as the second attack. Compounds that can prevent inflammation by targeting LPS have potential therapeutic clinical application. Epigallocatechin-3-gallate (EGCG) has potent hepatocyte-protective effect and mild anti-hepatitis virus function. Here, we investigated whether EGCG attenuated the severity of inflammatory response in LPS-stimulated L02 hepatocytes. L02 hepatocytes were pretreated with EGCG for 2 h, then stimulated by LPS at 250 ng/ml. The expression levels of chemokine regulated upon activation normal T-cell expressed and secreted (Rantes) and monocyte chemotactic protein-1 (MCP-1), pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interferon-γ, adhesion molecule intercellular adhesion molecule-1 (ICAM-1), oxidant stress molecules nitric oxide (NO), vascular endothelial growth factor (VEGF), and matrix metalloproteinase-2 (MMP-2) were tested by enzyme-linked immunosorbent assay. The expression of total extracellular signal-regulated kinase 1/2 (ERK1/2), phospho-ERK1/2 (p-ERK1/2), p-AKT, total p38, phospho-p38 (p-p38), total p65 and phospho-p65 (p-p65), IκBα, phospho-IκBα (p-IκBα) and TNF receptor associated factor 2 were tested by western blot analysis. Our results showed that pre-treatment with EGCG could significantly reduce the production of TNF-α, Rantes, MCP-1, ICAM-1, NO, VEGF, and MMP-2 in LPS-stimulated L02 hepatocytes in a dose-dependent manner. The effect of EGCG may be related to the inhibition of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways by down-regulation of p-IκBα, p65, p-p65, p-p38, p-ERK1/2, and p-AKT. These results indicate that EGCG suppresses LPS-induced inflammatory response and oxidant stress and exerts its hepatocyte-protective activity partially by inhibiting NF-κB and MAPK pathways.

  15. Activation of Wnt/β-catenin signalling via GSK3 inhibitors direct differentiation of human adipose stem cells into functional hepatocytes

    PubMed Central

    Huang, Jieqiong; Guo, Xinyue; Li, Weihong; Zhang, Haiyan

    2017-01-01

    The generation of hepatocytes that are derived from human adipose stem cells (hASCs) represents an alternative to human hepatocytes for individualized therapeutic and pharmaceutical applications. However, the mechanisms facilitating hepatocyte differentiation from hASCs are not well understood. Here, we show that upon exposure to glycogen synthase kinase 3 (GSK3) inhibitors alone, the expression of definitive endoderm specific genes GATA4, FOXA2, and SOX17 in hASCs significantly increased in a manner with activation of Wnt/β-catenin signalling. Down regulation of the β-catenin expression attenuates the effect of GSK3 inhibitors on the induction of these specific genes. The cells induced using GSK3 inhibitors were directed to differentiate synchronously into hepatocyte-like cells (HLCs) after further combinations of soluble factors by a reproducible three-stage method. Moreover, hASC-HLCs induced using GSK3 inhibitors possess low-density lipoprotein uptake, albumin secretion, and glycogen synthesis ability, express important drug-metabolizing cytochrome P450 (CYP450) enzymes, and demonstrate CYP450 activity. Therefore, our findings suggest that activation of Wnt/β-catenin signalling via GSK3 inhibitors in definitive endoderm specification may represent an important mechanism mediating hASCs differentiated to functional hepatocyte. Furthermore, development of similar compounds may be useful for robust, potentially scalable and cost-effective generation of functional hepatocytes for drug screening and predictive toxicology platforms. PMID:28094799

  16. In vitro cultivation and cryopreservation of duck embryonic hepatocytes.

    PubMed

    Schacke, M; Glück, B; Wutzler, P; Sauerbrei, A

    2009-04-01

    Hepatitis B-virucidal testing of biocides in quantitative suspension tests using duck hepatitis B virus (DHBV) requires primary duck embryonic hepatocytes for viral propagation. To improve the test system and availability of these cells, commercial culture plates with different growth surfaces were tested for cell cultivation and different approaches for cryopreservation of hepatocyte suspension were examined. After 12 days of culture, the largest amounts of hepatocytes were grown in CellBIND and TTP plates and CellBIND surface showed the lowest tendency of monolayer detachment nearly comparable with collagen 1-coated CELLCOAT plates. For cryopreservation of hepatocyte suspension, the use of growth medium supplemented with fetal calf serum (FCS) and dimethyl sulfoxide (ME(2)SO), FCS supplemented with ME(2)SO or cryosafe-1 as cryoprotective agents provided the highest rates of surviving cells after thawing. The freezing-thawing process did not significantly reduce the susceptibility of hepatocytes to infection with DHBV. In conclusion, plates without collagen 1 such as CellBIND are recommended for cultivation of primary duck embryonic hepatocytes in infectivity experiments of DHBV for virucidal testing of biocides. The use of cryopreserved hepatocytes is possible when freshly isolated cells from the liver of duck embryos are not available.

  17. New method of hepatocyte transplantation and extracorporeal liver support.

    PubMed Central

    Demetriou, A A; Whiting, J; Levenson, S M; Chowdhury, N R; Schechner, R; Michalski, S; Feldman, D; Chowdhury, J R

    1986-01-01

    A technique has been developed by the authors that allows hepatocyte attachment on collagen-coated microcarriers resulting in prolonged hepatocyte viability and function both in vivo and in vitro. Rat hepatocytes were obtained by portal vein collagenase perfusion. Intraperitoneally transplanted microcarrier-attached normal hepatocytes into congeneic Gunn rats were functioning 3-4 weeks later, as shown by the presence and persistence of conjugated bilirubin in recipient bile, sustained decrease in serum bilirubin, uptake of Tc99m-DESIDA, and morphologic criteria. Intraperitoneal transplantation of normal microcarrier-attached hepatocytes into genetically albumin deficient rats (NAR) resulted in marked increase in plasma albumin levels (6 days without and 21 days with Cyclosporin A immunosuppression). Microcarrier-attached hepatocytes transplanted after 2 weeks of storage at -80 C into congeneic Gunn rats were viable and functional as assessed by criteria outlined above. An extracorporeal liver perfusion system was developed using the microcarrier-attached hepatocytes that was capable of synthesizing and conjugating bilirubin and synthesizing liver-specific proteins. Images FIGS. 5A and B. FIG. 7. FIG. 8. FIG. 9. FIG. 12. PMID:3530153

  18. Hepatocyte transplantation in children with liver cell failure

    PubMed Central

    Hamooda, Mohamed

    2016-01-01

    Patients with hepatic failure and liver-based metabolic disorders require management which is both costly and complex. Hepatocyte transplantation has been very encouraging as an alternative to organ transplantation for liver disease treatment, and studies in rodents, show that transplants involving isolated liver cells can reverse hepatic failure, and correct various metabolic deficiencies of the liver. This 2016 review is based on a literature search using PubMed including original articles, reviews, cases and clinical guidelines. The search terms were “hepatocyte transplantation”, “liver transplantation”, “liver cell failure”, “metabolic liver disorders”, “orthotropic liver transplantation”, “hepatocytes” and “stem cell transplantation”. The goal of this review is to summarize the significance of hepatocyte transplantation, the sources of hepatocytes and the barriers of hepatocyte transplantation using a detailed review of literature. Our review shows that treatment of patients with liver disease by hepatocyte transplantation has expanded exponentially, especially for patients suffering from liver-based metabolic disorders. Once hepatocyte transplantation has been shown to effectively replace organ transplantation for a portion of patients with life-threatening liver metabolic diseases and those with liver failure it will make cell therapy effective and available for a broad population of patients with liver disorders. PMID:27957309

  19. Isolation of centrolobular and perilobular hepatocytes after phenobarbital treatment

    PubMed Central

    1975-01-01

    Daily phenobarbital (PB) injections, on 3-7 consecutive days, induce an intense proliferation of smooth endoplasmic reticulum (ER) associated with a decrease of the glucose-6-phosphatase activity. This situation first affects the centrolobular hepatocytes, enhancing the degree of liver lobule heterogeneity. This experimental model was used for isolation and further subfractionation of hepatocytes on Ficoll density gradients, as described in the preceding paper. Profiles of protein, DNA, RNA, glycogen, phosphorylase, and glucose-6-phosphatase were determined all along the gradient. Two liver cell populations were distinguished: (a) light hepatocytes (mean density 1.10) present the same morphological characteristics as centrolobular cells, i.e., an abundant smooth ER composed of tubular elements, numerous small mitochondria, and few glycogen particles; (b) heavy hepatocytes (mean density 1.14) are characterized by large and compact glycogen areas and prominent rough endoplasmic cisternae, as are the perilobular cells. After incubation in the Wachstein-Meisel medium, Centrolobular hepatocytes exhibit dispersed reaction sites of glucose-6-phosphatase activity, whereas perilobular cells present a continuous and intense reaction. Morphometric determinations were carried out for both cell populations. Centrolobular PB hepatocytes are considerably enlarged (mean diameter: 23.7 mum); perilobular hepatocytes have a significantly smaller mean diameter of 19.2 mum, which is close to the values of control liver cells. PMID:167029

  20. CCN1 expression in hepatocytes contributes to macrophage infiltration in nonalcoholic fatty liver disease in mice.

    PubMed

    Bian, Zhaolian; Peng, Yanshen; You, Zhengrui; Wang, Qixia; Miao, Qi; Liu, Yuan; Han, Xiaofeng; Qiu, Dekai; Li, Zhiping; Ma, Xiong

    2013-01-01

    Our objective was to investigate the potential roles of CCN1 in the inflammation and macrophage infiltration of nonalcoholic fatty liver disease (NAFLD). The regulation of hepatic CCN1 expression was investigated in vitro with murine primary hepatocytes treated with free fatty acids or lipopolysaccharide (LPS) and in vivo with high-fat (HF) diet-fed mice or ob/ob mice. CCN1 protein and a liver-specific CCN1 expression plasmid were administered to mice fed a normal diet (ND) or HF diet. Myeloid-derived macrophages and RAW264.7 cells were also treated with CCN1 in vitro to determine the chemotactic effects of CCN1 on macrophages. LPS treatment significantly increased hepatic CCN1 expression in HF diet-fed mice and ob/ob mice. LPS and FFAs induced CCN1 expression in primary murine hepatocytes in vitro through the TLR4/MyD88/AP-1 pathway. CCN1 protein and overexpression of CCN1 in the liver induced more severe hepatic inflammation and macrophage infiltrates in HF mice than in ND mice. CCN1 recruited macrophages through activation of the Mek/Erk signaling pathway in myeloid-derived macrophages and RAW264.7 cells in vitro. Endotoxin and FFA-induced CCN1 expression in hepatocytes is involved in the hepatic proinflammatory response and macrophage infiltration in murine NAFLD.

  1. Mutant IDH inhibits HNF-4α to block hepatocyte differentiation and promote biliary cancer.

    PubMed

    Saha, Supriya K; Parachoniak, Christine A; Ghanta, Krishna S; Fitamant, Julien; Ross, Kenneth N; Najem, Mortada S; Gurumurthy, Sushma; Akbay, Esra A; Sia, Daniela; Cornella, Helena; Miltiadous, Oriana; Walesky, Chad; Deshpande, Vikram; Zhu, Andrew X; Hezel, Aram F; Yen, Katharine E; Straley, Kimberly S; Travins, Jeremy; Popovici-Muller, Janeta; Gliser, Camelia; Ferrone, Cristina R; Apte, Udayan; Llovet, Josep M; Wong, Kwok-Kin; Ramaswamy, Sridhar; Bardeesy, Nabeel

    2014-09-04

    Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are among the most common genetic alterations in intrahepatic cholangiocarcinoma (IHCC), a deadly liver cancer. Mutant IDH proteins in IHCC and other malignancies acquire an abnormal enzymatic activity allowing them to convert α-ketoglutarate (αKG) to 2-hydroxyglutarate (2HG), which inhibits the activity of multiple αKG-dependent dioxygenases, and results in alterations in cell differentiation, survival, and extracellular matrix maturation. However, the molecular pathways by which IDH mutations lead to tumour formation remain unclear. Here we show that mutant IDH blocks liver progenitor cells from undergoing hepatocyte differentiation through the production of 2HG and suppression of HNF-4α, a master regulator of hepatocyte identity and quiescence. Correspondingly, genetically engineered mouse models expressing mutant IDH in the adult liver show an aberrant response to hepatic injury, characterized by HNF-4α silencing, impaired hepatocyte differentiation, and markedly elevated levels of cell proliferation. Moreover, IDH and Kras mutations, genetic alterations that co-exist in a subset of human IHCCs, cooperate to drive the expansion of liver progenitor cells, development of premalignant biliary lesions, and progression to metastatic IHCC. These studies provide a functional link between IDH mutations, hepatic cell fate, and IHCC pathogenesis, and present a novel genetically engineered mouse model of IDH-driven malignancy.

  2. Reprogramming of Mice Primary Hepatocytes into Insulin-Producing Cells by Transfection with Multicistronic Vectors

    PubMed Central

    Luo, Haizhao; Chen, Rongping; Yang, Rui; Liu, Yan; Chen, Youping; Chen, Hong

    2014-01-01

    The neogenesis of insulin-producing cells (IPCs) from non-beta-cells has emerged as a potential method for treating diabetes mellitus (DM). Many groups have documented that activation of pancreatic transcription factor(s) in hepatocytes can improve the hyperglycemia in diabetic mice. In the present study, we explored a novel protocol that reprogrammed primary hepatocytes into functional IPCs by using multicistronic vectors carrying pancreatic and duodenal homeobox-1 (Pdx1), neurogenin 3 (Ngn3), and v-musculoaponeurotic fibrosarcoma oncogene homolog A (MafA). These triple-transfected cells activated multiple beta-cell genes, synthesized and stored considerable amounts of insulin, and released the hormone in a glucose-regulated manner in vitro. Furthermore, when transplanted into streptozotocin-induced diabetic mice, the cells markedly ameliorated glucose tolerance. Our results indicated that ectopic expression of Pdx1, Ngn3, and MafA facilitated hepatocytes-to-IPCs reprogramming. This approach may offer opportunities for treatment of DM. PMID:25006589

  3. Mitochondrial oxidative stress-induced hepatocyte apoptosis reflects increased molybdenum intake in caprine.

    PubMed

    Zhuang, Yu; Liu, Ping; Wang, Liqi; Luo, Junrong; Zhang, Caiying; Guo, Xiaoquan; Hu, Guoliang; Cao, Huabin

    2016-03-01

    Molybdenum (Mo) is an essential trace element for animals and humans. However, the high dietary intake of Mo leads to disease conditions in heavy metal pollution areas. To the best of our knowledge, the effect of high levels of Mo on the apoptosis of hepatocyte in goats has not been investigated. Therefore, the aim of the present in vivo study was to investigate the impact of Mo on mitochondrial oxidative stress and apoptosis genes in the liver using real-time quantitative polymerase chain reaction (RT-qPCR) and transmission electron microscopy, respectively. Thirty-six healthy goats were randomly divided into three groups: two groups treated with ammonium molybdate [(NH4)6·Mo7O24·H2O] at 15 and 45 mg Mo kg(-1) BW, respectively, and a control group without treatment. Liver samples were collected from individual goats at different time intervals. The levels of oxidative stress in the mitochondrial membrane and expression of liver-related apoptosis genes, including Bcl-2, Cyt c, caspase-3, and Smac, were examined. The results demonstrated that the levels of superoxide dismutase (SOD) and catalase (CAT) expression were significantly down-regulated in liver cells, whereas malondialdehyde (MDA), nitric oxide (NO), and total nitric oxide synthase (T-NOS) expression was up-regulated (P < 0.01). The expression of Smac, Cyt c, and caspase-3 was significantly up-regulated, whereas Bcl-2 expression was down-regulated in liver cells (P < 0.01). In addition, histopathological examination revealed varying degrees of vacuolization, irregularity, nuclear fission, and mitochondrial swelling and high-density electrons in the cytoplasm of hepatocytes in groups treated with 15 and 45 mg Mo kg(-1) BW. Thus, these results suggested that high molybdenum induced hepatocyte apoptosis and might involve a mitochondrial pathway.

  4. Impact of IL-27 on hepatocyte antiviral gene expression and function

    PubMed Central

    2016-01-01

    Background: Interleukin (IL)-27 is a member of the IL-6/IL-12 family of cytokines. It is a potent cytokine, with potential antiviral impact, and has been shown to play a role in modulating functions of diverse cell types, including Th1, Th2, and NK and B cells, demonstrating both pro- and anti-inflammatory roles.  In hepatocytes, it is capable of inducing signal transducer and activator of transcription (STAT)1, STAT3 and interferon-stimulated genes. Methods: To address its role in viral hepatitis, the antiviral activity of IL-27 against hepatitis C virus (HCV) and hepatitis B virus (HBV) was tested in vitro using cell-culture-derived infectious HCV (HCVcc) cell culture system and the HepaRG HBV cell culture model. To further investigate the impact of IL-27 on hepatocytes, Huh7.5 cells were treated with IL-27 to analyse the differentially expressed genes by microarray analysis. Furthermore, by quantitative PCR, we analyzed the up-regulation of chemokine (CXCL)-10 in response to IL-27. Results: In both HCV and HBV infection models, we observed only a modest direct antiviral effect. Microarray analysis showed that the up-regulated genes mostly belonged to antigen presentation and DNA replication pathways, and involved strong up-regulation of CXCL-10, a gene associated with liver inflammation. Overall, gene set enrichment analysis showed a striking correlation of these genes with those up-regulated in response to related cytokines in diverse cell populations. Conclusion: Our data indicate that IL-27 can have a significant pro-inflammatory impact in vitro, although the direct antiviral effect is modest. It may have a potential impact on hepatocyte function, especially chemokine expression and antigen presentation. PMID:28058287

  5. Hepatocyte-like cells transdifferentiated from a pancreatic origin can support replication of hepatitis B virus.

    PubMed

    Wang, Robert Yung-Liang; Shen, Chia-Ning; Lin, Min-Hui; Tosh, David; Shih, Chiaho

    2005-10-01

    Recently, a rat pancreatic cell line (AR42J-B13) was shown to transdifferentiate to hepatocyte-like cells upon induction with dexamethasone (Dex). The aim of this study is to determine whether transdifferentiated hepatocytes can indeed function like bona fide liver cells and support replication of hepatotropic hepatitis B virus (HBV). We stably transfected AR42J-B13 cells with HBV DNA and examined the expression of hepatocyte markers and viral activities in control and transdifferentiated cells. A full spectrum of HBV replicative intermediates, including covalently closed circular DNA (cccDNA) and Dane particles, were detected only after induction with Dex and oncostatin M. Strikingly, the small envelope protein and RNA of HBV were increased by 40- to 100-fold upon induction. When HBV RNAs were examined by primer extension analysis, novel core- and precore-specific transcripts were induced by Dex which initiated at nucleotide (nt) 1820 and nt 1789, respectively. Most surprisingly, another species of core-specific RNA, which initiates at nt 1825, is always present at almost equal intensity before and after Dex treatment, a result consistent with Northern blot analysis. The fact that HBV core protein is dramatically produced only after transdifferentiation suggests the possibility of both transcriptional and translational regulation of HBV core antigen in HBV-transfected AR42J-B13 cells. Upon withdrawal of Dex, HBV replication and gene expression decreased rapidly-less than 50% of the cccDNA remained detectable in 1.5 days. Our studies demonstrate that the transdifferentiated AR42J-B13 cells can function like bona fide hepatocytes. This system offers a new opportunity for basic research of virus-host interactions and pancreatic transdifferentiation.

  6. Screening and identification of differentially expressed genes in goose hepatocytes exposed to free fatty acid.

    PubMed

    Pan, Zhixiong; Wang, Jiwen; Kang, Bo; Lu, Lizhi; Han, Chunchun; Tang, Hui; Li, Liang; Xu, Feng; Zhou, Zehui; Lv, Jia

    2010-12-15

    The overaccumulation of triglycerides in hepatocytes induces hepatic steatosis; however, little is known about the mechanism of goose hepatic steatosis. The aim of this study was to define an experimental model of hepatocellular steatosis with TG overaccumulation and minimal cytotoxicity, using a mixture of various proportions of oleate and palmitate free fatty acids (FFAs) to induce fat-overloading, then using suppressive subtractive hybridization and a quantitative PCR approach to identify genes with higher or lower expression levels after the treatment of cells with FFA mixtures. Overall, 502 differentially expressed clones, representing 21 novel genes and 87 known genes, were detected by SSH. Based on functional clustering, up- and down-regulated genes were mostly related to carbohydrate and lipid metabolism, enzyme activity and signal transduction. The expression of 20 selected clones involved with carbohydrate and lipid metabolism pathways was further studied by quantitative PCR. The data indicated that six clones similar to the genes ChREBP, FoxO1, apoB, IHPK2, KIF1B, and FSP27, which participate in de novo synthesis of fatty acid and secretion of very low density lipoproteins, had significantly lower expression levels in the hepatocytes treated with FFA mixtures. Meanwhile, 13 clones similar to the genes DGAT-1, ACSL1, DHRS7, PPARα, L-FABP, DGAT-2, PCK, ACSL3, CPT-1, A-FABP, PPARβ, MAT, and ALDOB had significantly higher expression levels in the hepatocytes treated with FFA mixtures. These results suggest that several metabolic pathways are altered in goose hepatocytes, which may be useful for further research into the molecular mechanism of goose hepatic steatosis.

  7. Dichloroacetate Stimulates Glycogen Accumulation in Primary Hepatocytes through an Insulin-Independent Mechanism

    SciTech Connect

    Lingohr, Melissa K.; Bull, Richard J.; Kato-Weinstein, Junko; Thrall, Brian D. )

    2002-01-01

    Dichloroacetate (DCA), a by-product of water chlorination, causes liver cancer in B6C3F1 mice. A hallmark response observed in mice exposed to carcinogenic doses of DCA is an accumulation of hepatic glycogen content. To distinguish whether the in vivo glycogenic effect of DCA was dependent on insulin and insulin signaling proteins, experiments were conducted in isolated hepatocytes where insulin concentrations could be controlled. In hepatocytes isolated from male B6C3F1 mice, DCA increased glycogen levels in a dose-related manner, independently of insulin. The accumulation of hepatocellular glycogen induced by DCA was not the result of decreased glycogenolysis, since DCA had no effect on the rate of glucagon-stimulated glycogen breakdown. Glycogen accumulation caused by DCA treatment was not hindered by inhibitors of extracellular-regulated protein kinase kinase (Erk1/2 kinase or MEK) or p70 kDa S6 protein kinase (p70(S6K)), but was completely blocked by the phosphatidylinositol 3-kinase (PI3K) inhibitors, LY294002 and wortmannin. Similarly, insulin-stimulated glycogen deposition was not influenced by the Erk1/2 kinase inhibitor, PD098509, or the p70(S6K) inhibitor, rapamycin. Unlike DCA-stimulated glycogen deposition, PI3K-inhibition only partially blocked the glycogenic effect of insulin. DCA did not cause phosphorylation of the downstream PI3K target protein, protein kinase B (PKB/Akt). The phosphorylation of PKB/Akt did not correlate to insulin-stimulated glycogenesis either. Similar to insulin, DCA in the medium decreased IR expression in isolated hepatocytes. The results indicate DCA increases hepatocellular glycogen accumulation through a PI3K-dependent mechanism that does not involve PKB/Akt and is, at least in part, different from the classical insulin-stimulated glycogenesis pathway. Somewhat surprisingly, insulin-stimulated glycogenesis also appears not to involve PKB/Akt in isolated murine hepatocytes.

  8. Comparative Transcriptomic Analysis of Primary Duck Hepatocytes Provides Insight into Differential Susceptibility to DHBV Infection

    PubMed Central

    Liu, Gang; Liu, Lei; Yu, Yao; Ding, Guohui; Zhao, Yanfeng; Li, Yixue; Xie, Youhua; Zhang, Junqi; Qu, Di

    2016-01-01

    Primary duck hepatocytes (PDH) displays differential susceptibility to duck hepatitis B virus when maintained in the media supplemented with fetal bovine serum or dimethyl sulfoxide (DMSO) which has been widely used for the maintenance of hepatocytes, and prolonging susceptibility to hepadnavirus. However the mechanism underlying maintenance of susceptibility to hepadnavirus by DMSO treatment remains unclear. In this study, a global transcriptome analysis of PDHs under different culture conditions was conducted for investigating the effects of DMSO on maintenance of susceptibility of PDH to DHBV in vitro. The 384 differential expressed genes (DEGs) were identified by comparisons between each library pair (PDHs cultured with or without DMSO or fresh isolated PDH). We analyzed canonical pathways in which the DEGs were enriched in Hepatic Fibrosis / Hepatic Stellate Cell Activation, Bile Acid Biosynthesis and Tight Junction signaling. After re-annotation against human genome data, the 384 DEGs were pooled together with proteins belonging to hepatitis B pathway to construct a protein-protein interaction network. The combination of decreased expression of liver-specific genes (CYP3A4, CYP1E1, CFI, RELN and GSTA1 et al) with increased expression of hepatocyte-dedifferentiation-associated genes (PLA2G4A and PLCG1) suggested that in vitro culture conditions results in the fading of hepatocyte phenotype in PDHs. The expression of seven DEGs associated with tight junction formation (JAM3, PPP2R2B, PRKAR1B, PPP2R2C, MAGI2, ACTA2 and ACTG2) was up-regulated after short-term culture in vitro, which was attenuated in the presence of DMSO. Those results could shed light on DHBV infection associated molecular events affected by DMSO. PMID:26900848

  9. Hepatocyte Hypoxia Inducible Factor-1 Mediates the Development of Liver Fibrosis in a Mouse Model of Nonalcoholic Fatty Liver Disease

    PubMed Central

    Mesarwi, Omar A.; Shin, Mi-Kyung; Bevans-Fonti, Shannon; Schlesinger, Christina; Shaw, Janet; Polotsky, Vsevolod Y.

    2016-01-01

    Background Obstructive sleep apnea (OSA) is associated with the progression of non-alcoholic fatty liver disease (NAFLD) to steatohepatitis and fibrosis. This progression correlates with the severity of OSA-associated hypoxia. In mice with diet induced obesity, hepatic steatosis leads to liver tissue hypoxia, which worsens with exposure to intermittent hypoxia. Emerging data has implicated hepatocyte cell signaling as an important factor in hepatic fibrogenesis. We hypothesized that hepatocyte specific knockout of the oxygen sensing α subunit of hypoxia inducible factor-1 (HIF-1), a master regulator of the global response to hypoxia, may be protective against the development of liver fibrosis. Methods Wild-type mice and mice with hepatocyte-specific HIF-1α knockout (Hif1a-/-hep) were fed a high trans-fat diet for six months, as a model of NAFLD. Hepatic fibrosis was evaluated by Sirius red stain and hydroxyproline assay. Liver enzymes, fasting insulin, and hepatic triglyceride content were also assessed. Hepatocytes were isolated from Hif1a-/-hep mice and wild-type controls and were exposed to sustained hypoxia (1% O2) or normoxia (16% O2) for 24 hours. The culture media was used to reconstitute type I collagen and the resulting matrices were examined for collagen cross-linking. Results Wild-type mice on a high trans-fat diet had 80% more hepatic collagen than Hif1a-/-hep mice (2.21 μg collagen/mg liver tissue, versus 1.23 μg collagen/mg liver tissue, p = 0.03), which was confirmed by Sirius red staining. Body weight, liver weight, mean hepatic triglyceride content, and fasting insulin were similar between groups. Culture media from wild-type mouse hepatocytes exposed to hypoxia allowed for avid collagen cross-linking, but very little cross-linking was seen when hepatocytes were exposed to normoxia, or when hepatocytes from Hif1a-/-hep mice were used in hypoxia or normoxia. Conclusions Hepatocyte HIF-1 mediates an increase in liver fibrosis in a mouse model of

  10. Hepatocytic parental progenitor cells of rat small hepatocytes maintain self-renewal capability after long-term culture

    PubMed Central

    Ishii, Masayuki; Kino, Junichi; Ichinohe, Norihisa; Tanimizu, Naoki; Ninomiya, Takafumi; Suzuki, Hiromu; Mizuguchi, Toru; Hirata, Koichi; Mitaka, Toshihiro

    2017-01-01

    The liver has a variety of functions for maintaining homeostasis, and hepatocytes play a major role. In contrast with the high regenerative capacity of mature hepatocytes (MHs) in vivo, they have not been successfully expanded ex vivo. Here we demonstrate that CD44-positive cells sorted from small hepatocyte (SH) colonies derived from a healthy adult rat liver can proliferate on a Matrigel-coated dish in serum-free chemically defined medium; in addition, a subpopulation of the cells can divide more than 50 times in a period of 17 weeks every 4-week-passage. The passage cells retained the capability to recover highly differentiated functions, such as glycogen storage, CYP activity and bile secretion. When Matrigel-treated cells from the third passage were transplanted into retrorsine/partial hepatectomy-treated rat livers, the cells engrafted to differentiate into MHs and cholangiocytes. These results suggest that long-term cultured CD44+ SHs retain hepatocytic characteristics in vitro and the capability to differentiate into hepatocytes and cholangiocytes in vivo. Thus, a newly identified subpopulation of MHs possessing the attributes of hepatocytic stem/progenitor cells can be passaged several times without losing hepatocytic characteristics.

  11. Hepatocyte growth factor in renal failure: promise and reality.

    PubMed

    Vargas, G A; Hoeflich, A; Jehle, P M

    2000-04-01

    Can science discover some secrets of Greek mythology? In the case of Prometheus, we can now suppose that his amazing hepatic regeneration was caused by a peptide growth factor called hepatocyte growth factor (HGF). Increasing evidence indicates that HGF acts as a multifunctional cytokine on different cell types. This review addresses the molecular mechanisms that are responsible for the pleiotropic effects of HGF. HGF binds with high affinity to its specific tyrosine kinase receptor c-met, thereby stimulating not only cell proliferation and differentiation, but also cell migration and tumorigenesis. The three fundamental principles of medicine-prevention, diagnosis, and therapy-may be benefited by the rational use of HGF. In renal tubular cells, HGF induces mitogenic and morphogenetic responses. In animal models of toxic or ischemic acute renal failure, HGF acts in a renotropic and nephroprotective manner. HGF expression is rapidly up-regulated in the remnant kidney of nephrectomized rats, inducing compensatory growth. In a mouse model of chronic renal disease, HGF inhibits the progression of tubulointerstitial fibrosis and kidney dysfunction. Increased HGF mRNA transcripts were detected in mesenchymal and tubular epithelial cells of rejecting kidney. In transplanted patients, elevated HGF levels may indicate renal rejection. When HGF is considered as a therapeutic agent in human medicine, for example, to stimulate kidney regeneration after acute injury, strategies need to be developed to stimulate cell regeneration and differentiation without an induction of tumorigenesis.

  12. Thyroid hormone and dehydroepiandrosterone permit gluconeogenic hormone responses in hepatocytes.

    PubMed

    Kneer, N; Lardy, H

    2000-03-01

    The importance of the sn-glycerol- 3-phosphate (G-3-P) electron transfer shuttle in hormonal regulation of gluconeogenesis was examined in hepatocytes from rats with decreased mitochondrial G-3-P dehydrogenase activity (thyroidectomized) or increased G-3-P dehydrogenase activity [triiodothyronine (T(3)) or dehydroepiandrosterone (DHEA) treated]. Rates of glucose formation from 10 mM lactate, 10 mM pyruvate, or 2.5 mM dihydroxyacetone were somewhat less in hypothyroid cells than in cells from normal rats but gluconeogenic responses to calcium addition and to norepinephrine (NE), glucagon (G), or vasopressin (VP) were similar to the responses observed in cells from normal rats. However, with 2. 5 mM glycerol or 2.5 mM sorbitol, substrates that must be oxidized in the cytosol before conversion to glucose, basal gluconeogenesis was not appreciably altered by hypothyroidism but responses to calcium and to the calcium-mobilizing hormones were abolished. Injecting thyroidectomized rats with T(3) 2 days before preparing the hepatocytes greatly enhanced gluconeogenesis from glyc erol and restored the response to Ca(2+) and gluconeogenic hormones. Feeding dehydroepiandrosterone for 6 days depressed gluconeogenesis from lactate or pyruvate but substantially increased glucose production from glycerol in euthyroid cells and restored responses to Ca(2+) in hypothyroid cells metabolizing glycerol. Euthyroid cells metabolizing glycerol or sorbitol use the G-3-P and malate/aspartate shuttles to oxidize excess NADH generated in the cytosol. The transaminase inhibitor aminooxyacetate (AOA) decreased gluconeogenesis from glycerol 40%, but had little effect on responses to Ca(2+) and NE. However, in hypothyroid cells, with minimal G-3-P dehydrogenase, AOA decreased gluconeogenesis from glycerol more than 90%. Thus, the basal rate of gluconeogenesis from glycerol in the euthyroid cells is only partly dependent on electron transport from cytosol to mitochondria via the malate

  13. Coculture and Long-Term Maintenance of Hepatocytes.

    PubMed

    Cohen, Merav; Levy, Gahl; Nahmias, Yaakov

    2015-01-01

    The liver is the largest internal organ in mammals, serving a wide spectrum of vital functions. Loss of liver function due to drug toxicity, progressive fatty liver disease, or viral infection is a major cause of death in the United States of America. Pharmaceutical and cosmetic toxicity screening, basic research and the development of bioartificial liver devices require long-term hepatocyte culture techniques that sustain hepatocyte morphology and function. In recent years, several techniques have been developed that can support high levels of liver-specific gene expression, metabolic function, and synthetic activity for several weeks in culture. These include the collagen double gel configuration, hepatocyte spheroids, coculture with nonparenchymal cells, and micropatterned cocultures. This chapter will cover the current status of hepatocyte culture techniques, including media formulation, oxygen supply, and heterotypic cell-cell interactions.

  14. Direct reprogramming of human fibroblasts to functional and expandable hepatocytes.

    PubMed

    Huang, Pengyu; Zhang, Ludi; Gao, Yimeng; He, Zhiying; Yao, Dan; Wu, Zhitao; Cen, Jin; Chen, Xiaotao; Liu, Changcheng; Hu, Yiping; Lai, Dongmei; Hu, Zhenlei; Chen, Li; Zhang, Ying; Cheng, Xin; Ma, Xiaojun; Pan, Guoyu; Wang, Xin; Hui, Lijian

    2014-03-06

    The generation of large numbers of functional human hepatocytes for cell-based approaches to liver disease is an important and unmet goal. Direct reprogramming of fibroblasts to hepatic lineages could offer a solution to this problem but so far has only been achieved with mouse cells. Here, we generated human induced hepatocytes (hiHeps) from fibroblasts by lentiviral expression of FOXA3, HNF1A, and HNF4A. hiHeps express hepatic gene programs, can be expanded in vitro, and display functions characteristic of mature hepatocytes, including cytochrome P450 enzyme activity and biliary drug clearance. Upon transplantation into mice with concanavalin-A-induced acute liver failure and fatal metabolic liver disease due to fumarylacetoacetate dehydrolase (Fah) deficiency, hiHeps restore the liver function and prolong survival. Collectively, our results demonstrate successful lineage conversion of nonhepatic human cells into mature hepatocytes with potential for biomedical and pharmaceutical applications.

  15. Toxicity of ethacrynic acid in isolated rat hepatocytes.

    PubMed

    Yamamoto, K; Masubuchi, Y; Narimatsu, S; Kobayashi, S; Horie, T

    2002-04-01

    Ethacrynic acid, a loop diuretic drug, caused lipid peroxidation in isolated rat hepatocytes. The thiobarbituric acid reactive substances (TBARS) formation showed a good correlation with the leakage of glutamic-oxaloacetic acid transaminase (GOT) from the hepatocytes. The addition of antioxidants such as N, N'-diphenyl-p-phenylenediamine (DPPD) and promethazine to the isolated rat hepatocyte suspension containing ethacrynic acid prevented the lipid peroxidation and decreased the GOT leakage to some extent. SKF-525A inhibited the oxidative metabolism of ethacrynic acid and decreased the TBARS formation, suggesting that the lipid peroxidation was caused by the oxidative metabolism. The intracellular reduced glutathione markedly decreased in the hepatocyte suspension containing ethacrynic acid and the hepatocellular protein sulfhydryls were decreased, which was negatively correlated with the GOT leakage. Thus the ethacrynic acid-induced hepatotoxicity was found to be related to the lipid peroxidation and the decrease of cellular protein sulfhydryls.

  16. Heat-shock protein 90 alpha (HSP90α) modulates signaling pathways towards tolerance of oxidative stress and enhanced survival of hepatocytes of Mugil cephalus.

    PubMed

    Padmini, Ekambaram; Usha Rani, Munuswamy

    2011-07-01

    Oxidative stress causes damage at the cellular level and activates a number of signaling pathways. Earlier, we have demonstrated that pollutant-related oxidative stress upregulates heat-shock protein 90 alpha (HSP90α) against stress insult in hepatocytes of Mugil cephalus living in a polluted estuary. However, the impact of pollution-induced HSP90α upregulation on stress tolerance is not clear. Here we propose that the effect of stress resistance depends on the ability of HSP90α to modulate the signaling pathways involving proteins such as apoptosis signal-regulating kinase 1, c-Jun NH(2)-terminal protein kinase 1/2, signal transducers and activators of transcription, extracellular signal-regulated kinase 1/2, protein kinase B, nuclear factor-kappa binding, Ets-like protein 1, and B cell lymphoma-2. In order to investigate this, the activation of HSP90α-associated signaling molecules was examined by Western blotting and immunohistochemistry. The relationship between the protein expression patterns was identified by Spearman's rank correlation analysis. The signaling proteins exhibited differential modulation as revealed from their expression patterns in pollutant-exposed fish hepatocytes, in comparison with the control fish hepatocytes. The results suggested that in spite of the prevalence of oxidative stress in pollutant-exposed fish hepatocytes, the stress-mediated induction of HSP90α enabled the hepatocytes to become stress tolerant and to survive by modulating the actions of key proteins and kinases in the signal transduction pathways.

  17. cAMP prevents TNF-induced apoptosis through inhibiting DISC complex formation in rat hepatocytes.

    PubMed

    Bhattacharjee, Rajesh; Xiang, Wenpei; Wang, Yinna; Zhang, Xiaoying; Billiar, Timothy R

    2012-06-22

    Tumor necrosis factor α (TNF) is a pleiotropic proinflammatory cytokine that plays a role in immunity and the control of cell proliferation, cell differentiation, and apoptosis. The pleiotropic nature of TNF is due to the formation of different signaling complexes upon the binding of TNF to its receptor, TNF receptor type 1 (TNFR1). TNF induces apoptosis in various mammalian cells when the cells are co-treated with a transcription inhibitor like actinomycin D (ActD). When TNFR1 is activated, it recruits an adaptor protein, TNF receptor-associated protein with death domain (TRADD), through its cytoplasmic death effector domain (DED). TRADD, in turn, recruits other signaling proteins, including TNF receptor-associated protein 2 (TRAF2) and receptor-associated protein kinase (RIPK) 1, to form a complex. Subsequently, this complex combines with FADD and procaspase-8, converts into a death-inducing signaling complex (DISC) to induce apoptosis. Cyclic AMP (cAMP) is a second messenger that regulates various cellular processes such as cell proliferation, gene expression, and apoptosis. cAMP analogues are reported to act as anti-apoptotic agents in various cell types, including hepatocytes. We found that a cAMP analogue, dibutyryl cAMP (db-cAMP), inhibits TNF+ActD-induced apoptosis in rat hepatocytes. The protein kinase A (PKA) inhibitor KT-5720 reverses this inhibitory effect of cAMP on apoptosis. Cytoprotection by cAMP involves down-regulation of various apoptotic signal regulators like TRADD and FADD and inhibition of caspase-8 and caspase-3 cleavage. We also found that cAMP exerts its affect at the proximal level of TNF signaling by inhibiting the formation of the DISC complex upon the binding of TNF to TNFR1. In conclusion, our study shows that cAMP prevents TNF+ActD-induced apoptosis in rat hepatocytes by inhibiting DISC complex formation.

  18. Tanshinone I increases CYP1A2 protein expression and enzyme activity in primary rat hepatocytes.

    PubMed

    Lee, Wayne Y W; Zhou, Xuelin; Or, Penelope M Y; Kwan, Yiu Wa; Yeung, John H K

    2012-01-15

    This study investigated the effects of Danshen and its active ingredients on the protein expression and enzymatic activity of CYP1A2 in primary rat hepatocytes. The ethanolic extract of Danshen roots (containing mainly tanshinones) inhibited CYP1A2-catalyzed phenacetin O-deethylation (IC(50)=24.6 μg/ml) in primary rat hepatocytes while the water extract containing mainly salvianolic acid B and danshenshu had no effect. Individual tanshinones such as cryptotanshinone, dihydrotanshinone, tanshinone IIA inhibited the CYP1A2-mediated metabolism with IC(50) values at 12.9, 17.4 and 31.9 μM, respectively. After 4-day treatment of the rat hepatocytes, the ethanolic extract of Danshen and tanshinone I increased rat CYP1A2 activity by 6.8- and 5.2-fold, respectively, with a concomitant up-regulation of CYP1A2 protein level by 13.5- and 6.5-fold, respectively. CYP1A2 induction correlated with the up-regulation of mRNA level of aryl hydrocarbon receptor (AhR), which suggested a positive feedback mechanism of tanshinone I-mediated CYP1A2 induction. A formulated Danshen pill (containing mainly danshensu and salvianolic acid B and the tanshinones) up-regulated CYP1A2 protein expression and enzyme activity, but danshensu and salvianolic acid B, when used individually, did not affect CYP1A2 activity. This study was the first report on the Janus action of the tanshinones on rat CYP1A2 activity.

  19. Integrated analysis of gene expression and DNA methylation changes induced by hepatocyte growth factor in human hepatocytes.

    PubMed

    Xie, Cheng-Rong; Sun, Hongguang; Wang, Fu-Qiang; Li, Zhao; Yin, Yi-Rui; Fang, Qin-Liang; Sun, Yu; Zhao, Wen-Xiu; Zhang, Sheng; Zhao, Wen-Xing; Wang, Xiao-Min; Yin, Zhen-Yu

    2015-09-01

    Hepatocellular carcinoma (HCC) is the one of most common malignant tumors. The tumor microenvironment has a role in not only supporting growth and survival of tumor cells, but also triggering tumor recurrence and metastasis. Hepatocyte growth factor (HGF), one of the important growth factors in the tumor microenvironment, has an important role in angiogenesis, tumorigenesis and regeneration. However, the exact mechanism by which HGF regulates HCC initiation and development via epigenetic reprogramming has remained elusive. The present study focused on the epigenetic modification and target tumor-suppressive genes of HGF treatment in HCC. Expression profiling and DNA methylation array were performed to investigate the function of HGF and examine global genomic DNA methylation changes, respectively. Integrated analysis of gene expression and DNA methylation revealed potential tumor suppressor genes (TSGs) in HCC. The present study showed the multiple functions of HGF in tumorous and non‑tumorous pathways and global genomic DNA methylation changes. HGF treatment upregulated the expression of DNA methyltransferase 1 (DNMT1). Overexpression of DNMT1 in HCC patients correlated with the malignant potential and poor prognosis of HCC. Furthermore, integration analysis of gene expression and DNA methylation changes revealed novel potential tumor suppressor genes TSGs including MYOCD, PANX2 and LHX9. The present study has provided mechanistic insight into epigenetic repression of TSGs through HGF‑induced DNA hypermethylation.

  20. Par1b Induces Asymmetric Inheritance of Plasma Membrane Domains via LGN-Dependent Mitotic Spindle Orientation in Proliferating Hepatocytes

    PubMed Central

    Slim, Christiaan L.; Lázaro-Diéguez, Francisco; Bijlard, Marjolein; Toussaint, Mathilda J. M.; de Bruin, Alain; Du, Quansheng; Müsch, Anne; van IJzendoorn, Sven C. D.

    2013-01-01

    The development and maintenance of polarized epithelial tissue requires a tightly controlled orientation of mitotic cell division relative to the apical polarity axis. Hepatocytes display a unique polarized architecture. We demonstrate that mitotic hepatocytes asymmetrically segregate their apical plasma membrane domain to the nascent daughter cells. The non-polarized nascent daughter cell can form a de novo apical domain with its new neighbor. This asymmetric segregation of apical domains is facilitated by a geometrically distinct “apicolateral” subdomain of the lateral surface present in hepatocytes. The polarity protein partitioning-defective 1/microtubule-affinity regulating kinase 2 (Par1b/MARK2) translates this positional landmark to cortical polarity by promoting the apicolateral accumulation of Leu-Gly-Asn repeat-enriched protein (LGN) and the capture of nuclear mitotic apparatus protein (NuMA)–positive astral microtubules to orientate the mitotic spindle. Proliferating hepatocytes thus display an asymmetric inheritance of their apical domains via a mechanism that involves Par1b and LGN, which we postulate serves the unique tissue architecture of the developing liver parenchyma. PMID:24358023

  1. Protective Role of Morin, a Flavonoid, against High Glucose Induced Oxidative Stress Mediated Apoptosis in Primary Rat Hepatocytes

    PubMed Central

    Kapoor, Radhika; Kakkar, Poonam

    2012-01-01

    Apoptosis is an early event of liver damage in diabetes and oxidative stress has been linked to accelerate the apoptosis in hepatocytes. Therefore, the compounds that can scavenge ROS may confer regulatory effects on high-glucose induced apoptosis. In the present study, primary rat hepatocytes were exposed to high concentration (40 mM) of glucose. At this concentration decreased cell viability and enhanced ROS generation was observed. Depleted antioxidant status of hepatocytes under high glucose stress was also observed as evident from transcriptional level and activities of antioxidant enzymes. Further, mitochondrial depolarisation was accompanied by the loss of mitochondrial integrity and altered expression of Bax and Bcl-2. Increased translocation of apoptotic proteins like AIF (Apoptosis inducing factor) & Endo-G (endonuclease-G) from its resident place mitochondria to nucleus was also observed. Cyt-c residing in the inter-membrane space of mitochondria also translocated to cytoplasm. These apoptotic proteins initiated caspase activation, DNA fragmentation, chromatin condensation, increased apoptotic DNA content in glucose treated hepatocytes, suggesting mitochondria mediated apoptotic mode of cell death. Morin, a dietary flavonoid from Psidium guajava was effective in increasing the cell viability and decreasing the ROS level. It maintained mitochondrial integrity, inhibited release of apoptotic proteins from mitochondria, prevented DNA fragmentation, chromatin condensation and hypodiploid DNA upon exposure to high glucose. This study confirms the capacity of dietary flavonoid Morin in regulating apoptosis induced by high glucose via mitochondrial mediated pathway through intervention of oxidative stress. PMID:22899998

  2. Intracytoplasmic Crystalline Inclusions in the Hepatocytes of an Antelope

    DTIC Science & Technology

    2010-01-01

    hepatocytes of a 13-year-old female Thomson’s gazelle . Histologically, multifocal to coalescing areas of many hepatocytes contained large cytoplasmic...hepatocellular crystalline inclusions in a gazelle . 2. Case Description A 13-year-old female Thomson’s gazelle (Eudorcas thomsoni) from a zoo was presented to...dead and had a history of severe parasitism. Postmortem examination revealed sparse body fat store in the gazelle . There was bilateral enlargement of

  3. Salvianolate Protects Hepatocytes from Oxidative Stress by Attenuating Mitochondrial Injury

    PubMed Central

    Zhao, Qiang; Peng, Yuan; Huang, Kai; Lei, Yang; Liu, Hong-Liang; Tao, Yan-Yan

    2016-01-01

    Salvianolate is widely used to treat angiocardiopathy in clinic in China, but its application in liver diseases remains unclear. Our study aims to investigate the effect of Salvianolate on rat hepatic injury by protecting hepatocyte mitochondria. To evaluate the effects of Salvianolate on injured hepatocytes, alpha mouse liver 12 (AML-12) cells were induced with hydrogen peroxide (H2O2) and treated with Salvianolate. Cell viability and MitoTracker Green for mitochondria and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazole-carbocyanide iodine (JC-1) levels and cytochrome C (Cyto-C) expressions were detected in vitro. To identify the effect of Salvianolate on protecting against mitochondria injury, male Wistar rats were injected with carbon tetrachloride (CCl4) and treated with Salvianolate (40 mg·kg−1). Serum liver function, parameters for peroxidative damage, hematoxylin and eosin (H&E) staining, and transmission electron microscope (TEM) of hepatocyte mitochondria were assayed. Our results showed that Salvianolate effectively protected hepatocytes, increased mitochondria vitality, and decreased Cyto-C expressions in vitro. Besides, Salvianolate alleviated the liver function, attenuated the indicators of peroxidation, and relieved the mitochondria injury in vivo. In conclusion, Salvianolate is effective in protecting hepatocytes from injury in vitro and in vivo, and the mechanism might be related to its protective effect on hepatocyte mitochondria against oxidative stress. PMID:27340417

  4. Hepatocyte apoptosis in dairy cattle during the transition period.

    PubMed

    Tharwat, Mohamed; Takamizawa, Aya; Hosaka, Yoshinao Z; Endoh, Daiji; Oikawa, Shin

    2012-10-01

    The objective of this study was to investigate hepatocyte apoptosis in dairy cows during the transition period. Four clinically healthy, pregnant dairy cattle were used. The cows had no clinical diseases throughout this study. Blood samples were collected and livers were biopsied from the cows at 3 different times: 3 weeks before expected partition (wk -3); during parturition (wk 0), and 3 weeks (wk +3) after parturition. The damage to deoxyribonucleic acid (DNA) caused by hepatocytes was evaluated by comet assay. The apoptotic features of hepatocytes were examined by immunohistochemistry and electron microscopic analyses. The hepatic triglyceride content markedly increased at wk 0 and wk +3 compared with the values at wk -3. The results of the comet assay showed increases in the mean tail moment values of hepatic cells after parturition in all cows, which suggested increased DNA damage. Histopathologically, the hepatocytes began to contain lipid droplets at wk 0 and were severely opacified at wk +3. Caspase-3-positive and single-stranded DNA-(ssDNA)-positive cells were first detected in the liver after parturition. Condensation of nuclear chromatin, a typical sign of apoptosis, was confirmed by transmission electron microscopy after parturition. These results suggest that apoptosis is induced in hepatocytes of dairy cows around parturition and may result from lipotoxicity in hepatocytes.

  5. Induction of hepatocyte proliferation by retinoic acid.

    PubMed

    Ledda-Columbano, G M; Pibiri, M; Molotzu, F; Cossu, C; Sanna, L; Simbula, G; Perra, A; Columbano, A

    2004-11-01

    Retinoids have been shown to exert an anticarcinogenic effect through suppression of the cell cycle, induction of apoptosis and/or differentiation. In rat liver, in particular, retinoic acid has been shown to inhibit regeneration after partial hepatectomy, most probably through repression of the expression of c-fos and c-jun. Surprisingly enough, in spite of the proposed therapeutic effects of all-trans retinoic acid (tRA) no data are available on its effect on normal adult liver. Here, we show that tRA administration in the diet (150 mg/kg) increased DNA synthesis in mouse liver, at 1 and 2 weeks, with a return to control values at 4 weeks (labelling index was 16.5, 8.3 and 3.3%, respectively, versus control values of 1.4, 1.3 and 2.5%). Increase in mitotic index paralleled that of bromodeoxyuridine incorporation. Kinetic studies showed that entry into S phase began between 24 and 48 h, with a peak between 96 and 120 h. Histological observation of the liver and biochemical evaluation of the levels of serum glutamate-pyruvate transaminases did not reveal any evidence of cell death demonstrating that increased DNA synthesis was not due to tRA-induced liver damage and regeneration, but rather the consequence of a direct mitogenic effect. In addition, analysis of total hepatic DNA content after a 7-day treatment showed a significant increase in tRA-fed mice compared with controls (21.11 mg/100 g body wt in tRA-fed mice versus 15.67 mg/100 g body wt of controls). Hepatocyte proliferation in tRA-fed mice was associated with increased hepatic levels of cyclin D1, E and A, and enhanced expression of the member of pRb family, p107. In conclusion, the results showed that tRA induces hepatocyte proliferation in the absence of cell death, similarly to other ligands of steroid/thyroid hormone nuclear receptor superfamily. The mitogenic effect of tRA cautions about its possible use for antitumoral purposes in liver carcinogenesis.

  6. The antioxidant action and mechanism of selenizing Schisandra chinensis polysaccharide in chicken embryo hepatocyte.

    PubMed

    Yue, Chanjuan; Chen, Jin; Hou, Ranran; Tian, Weijun; Liu, Kuanhui; Wang, Deyun; Lu, Yu; Liu, Jiaguo; Wu, Yi; Hu, Yuanliang

    2017-05-01

    The antioxidant action and mechanism of selenizing schisandra chinensis polysaccharide (sSCP) were investigated in chicken embryo hepatocyte (CEH) taking schisandra chinensis polysaccharide (SCP) and N-acetyl-L-cysteine (NAC) as control. The CEH was cultured and treated with sSCP, then exposed to H2O2. The CEHs' viability, apoptosis, ROS and antioxidase contents and the protein expression in MAPKs pathway and mitochondrion-dependence apoptotic signal pathway were assayed. The results showed that sSCP could significantly raise the cell viability and the activities of SOD, CAT and GSH-Px, decrease the cell apoptosis and the content of LDH, AST, ALT and MDA, down-regulate the protein expression of p-JNK1, p-ERK1/2, p-p38, Bax, Caspase 3 and cytochrome C, and up-regulate the protein expression of Bcl-2 in comparison with H2O2 control group. The action of sSCP were stronger than those of SCP and NAC. These results indicated that selenylation modification could significantly enhance the antioxidant activity of SCP, sSCP could significantly protect hepatocyte from oxidative damage induced by H2O2, and its mechanism was by regulating the protein expression in MAPKs and mitochondrion-dependence apoptotic signaling pathways.

  7. AMP-activated protein kinase counteracted the inhibitory effect of glucose on the phosphoenolpyruvate carboxykinase gene expression in rat hepatocytes.

    PubMed

    Hubert, A; Husson, A; Chédeville, A; Lavoinne, A

    2000-09-22

    The effect of AMP-activated protein kinase (AMPK) in the regulation of the phosphoenolpyruvate carboxykinase (PEPCK) gene expression was studied in isolated rat hepatocytes. Activation of AMPK by AICAR counteracted the inhibitory effect of glucose on the PEPCK gene expression, both at the mRNA and the transcriptional levels. It is proposed that a target for AMPK is involved in the inhibitory effect of glucose on PEPCK gene transcription.

  8. Nonalcoholic Lipid Accumulation and Hepatocyte Malignant Transformation

    PubMed Central

    Gu, Juanjuan; Yao, Min; Yao, Dengbing; Wang, Li; Yang, Xuli; Yao, Dengfu

    2016-01-01

    Abstract Worldwide incidence of hepatocellular carcinoma (HCC) is steadily increasing, highlighting its status as a public health concern, particularly due to its significant association with other comorbidities, such as diabetes. However, nonalcoholic fatty liver disease (NAFLD) has emerged as a primary risk factor, with its own prevalence increasing in recent years, and it has gradually caught up with the historical primary etiological factors of infection with hepatitis B virus and hepatitis C virus, exposure to aflatoxin, or alcohol liver disease. The deeply worrisome aspects of all of these high risk factors, however, are their remarkable presence within populations. Systemic and genetic mechanisms involved in the malignant transformation of liver cells, as well as useful biomarkers of early stage HCC are being investigated. However, the exact mechanisms underlying the interrelation of NAFLD and HCC remain largely unknown. In this review, some of the recent advances in our understanding of liver lipid accumulation are summarized and discussed to provide insights into the relationship between NAFLD and hepatocyte malignant transformation. PMID:27350942

  9. Interference of CuO nanoparticles with metal homeostasis in hepatocytes under sub-toxic conditions

    NASA Astrophysics Data System (ADS)

    Cuillel, Martine; Chevallet, Mireille; Charbonnier, Peggy; Fauquant, Caroline; Pignot-Paintrand, Isabelle; Arnaud, Josiane; Cassio, Doris; Michaud-Soret, Isabelle; Mintz, Elisabeth

    2014-01-01

    Copper oxide nanoparticles (CuO-NP) were studied for their toxicity and mechanism of action on hepatocytes (HepG2), in relation to Cu homeostasis disruption. Indeed, hepatocytes, in the liver, are responsible for the whole body Cu balance and should be a major line of defence in the case of exposure to CuO-NP. We investigated the early responses to sub-toxic doses of CuO-NP and compared them to equivalent doses of Cu added as salt to see if there is a specific nano-effect related to Cu homeostasis in hepatocytes. The expression of the genes encoding the Cu-ATPase ATP7B, metallothionein 1X, heme oxygenase 1, heat shock protein 70, superoxide dismutase 1, glutamate cysteine ligase modifier subunit, metal responsive element-binding transcription factor 1 and zinc transporter 1 was analyzed by qRT-PCR. These genes are known to be involved in response to Cu, Zn and/or oxidative stresses. Except for MTF1, ATP7B and SOD1, we clearly observed an up regulation of these genes expression in CuO-NP treated cells, as compared to CuCl2. In addition, ATP7B trafficking from the Golgi network to the bile canaliculus membrane was observed in WIF-B9 cells, showing a need for Cu detoxification. This shows an increase in the intracellular Cu concentration, probably due to Cu release from endosomal CuO-NP solubilisation. Our data show that CuO-NP enter hepatic cells, most probably by endocytosis, bypassing the cellular defence mechanism against Cu, thus acting as a Trojan horse. Altogether, this study suggests that sub-toxic CuO-NP treatments induce successively a Cu overload, a Cu-Zn exchange on metallothioneins and MTF1 regulation on both Cu and Zn homeostasis.Copper oxide nanoparticles (CuO-NP) were studied for their toxicity and mechanism of action on hepatocytes (HepG2), in relation to Cu homeostasis disruption. Indeed, hepatocytes, in the liver, are responsible for the whole body Cu balance and should be a major line of defence in the case of exposure to CuO-NP. We investigated

  10. cAMP prevents TNF-induced apoptosis through inhibiting DISC complex formation in rat hepatocytes

    SciTech Connect

    Bhattacharjee, Rajesh; Xiang, Wenpei; Wang, Yinna; Zhang, Xiaoying

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer cAMP blocks cell death induced by TNF and actinomycin D in cultured hepatocytes. Black-Right-Pointing-Pointer cAMP blocks NF-{kappa}B activation induced by TNF and actinomycin D. Black-Right-Pointing-Pointer cAMP blocks DISC formation following TNF and actinomycin D exposure. Black-Right-Pointing-Pointer cAMP blocks TNF signaling at a proximal step. -- Abstract: Tumor necrosis factor {alpha} (TNF) is a pleiotropic proinflammatory cytokine that plays a role in immunity and the control of cell proliferation, cell differentiation, and apoptosis. The pleiotropic nature of TNF is due to the formation of different signaling complexes upon the binding of TNF to its receptor, TNF receptor type 1 (TNFR1). TNF induces apoptosis in various mammalian cells when the cells are co-treated with a transcription inhibitor like actinomycin D (ActD). When TNFR1 is activated, it recruits an adaptor protein, TNF receptor-associated protein with death domain (TRADD), through its cytoplasmic death effector domain (DED). TRADD, in turn, recruits other signaling proteins, including TNF receptor-associated protein 2 (TRAF2) and receptor-associated protein kinase (RIPK) 1, to form a complex. Subsequently, this complex combines with FADD and procaspase-8, converts into a death-inducing signaling complex (DISC) to induce apoptosis. Cyclic AMP (cAMP) is a second messenger that regulates various cellular processes such as cell proliferation, gene expression, and apoptosis. cAMP analogues are reported to act as anti-apoptotic agents in various cell types, including hepatocytes. We found that a cAMP analogue, dibutyryl cAMP (db-cAMP), inhibits TNF + ActD-induced apoptosis in rat hepatocytes. The protein kinase A (PKA) inhibitor KT-5720 reverses this inhibitory effect of cAMP on apoptosis. Cytoprotection by cAMP involves down-regulation of various apoptotic signal regulators like TRADD and FADD and inhibition of caspase-8 and caspase-3 cleavage. We also found

  11. Early Results from the HexPak and GradPak Variable-Scale Dual-Head IFUs on the WIYN 3.5-meter Telescope

    NASA Astrophysics Data System (ADS)

    Hooper, Eric; Bershady, Matthew A.; Eigenbrot, Arthur; Wood, Corey M.; Buckley, Scott; Smith, Michael; Corson, Charles; Wolf, Marsha J.; Zhu, Guanying Y.; Vang, Andrea; Gallagher, John S.; Sheinis, Andrew; Washburn Astronomical Laboratories

    2015-01-01

    The WIYN Observatory recently installed two new integral field units (IFUs) on its 3.5-meter telescope on Kitt Peak, Arizona. Each IFU is unique in that it contains different sized fibers in the same head to optimize the tradeoff between spatial resolution and surface brightness sensitivity for observations of galaxies. These instruments were designed and constructed (M. Bershady, PI) at the University of Wisconsin's Washburn Astronomical Laboratory. HexPak, with a central core of 1 arcsec fibers surrounded by a halo of 3 arcsec fibers, was designed for early type, face-on disk, and quasar host galaxies. GradPak, with a series of rows of fibers of increasing diameter from 2 arcsec to 6 arcsec (5 different diameters total), was designed for edge-on galaxies, where the small fibers lie along the midplane and larger fibers sample the progressively lower surface brightnesses above the plane. The instruments were installed alongside the existing SparsePak IFU in late 2013 and have been used in several observing runs since. The different fiber sizes present additional data reduction challenges, particularly regarding flux calibration and sky subtraction. Early results on studies of the stellar populations of galaxies are quite promising and demonstrate the advantages of fiber sizes tailored to the objects under study. HexPak and GradPak were built with funds from NSF award ATI-0804576.

  12. Hepatic effects of a methionine-choline-deficient diet in hepatocyte RXR{alpha}-null mice

    SciTech Connect

    Gyamfi, Maxwell Afari; Tanaka, Yuji; He Lin; Klaassen, Curtis D.; Wan, Y.-J.Y.

    2009-01-15

    Retinoid X receptor-{alpha} (RXR{alpha}) is an obligate partner for several nuclear hormone receptors that regulate important physiological processes in the liver. In this study the impact of hepatocyte RXR{alpha} deficiency on methionine and choline deficient (MCD) diet-induced steatosis, oxidative stress, inflammation, and hepatic transporters gene expression were examined. The mRNA of sterol regulatory element-binding protein (SREBP)-regulated genes, important for lipid synthesis, were not altered in wild type (WT) mice, but were increased 2.0- to 5.4-fold in hepatocyte RXR{alpha}-null (H-RXR{alpha}-null) mice fed a MCD diet for 14 days. Furthermore, hepatic mRNAs and proteins essential for fatty acid {beta}-oxidation were not altered in WT mice, but were decreased in the MCD diet-fed H-RXR{alpha}-null mice, resulting in increased hepatic free fatty acid levels. Cyp2e1 enzyme activity and lipid peroxide levels were induced only in MCD-fed WT mice. In contrast, hepatic mRNA levels of pro-inflammatory factors were increased only in H-RXR{alpha}-null mice fed the MCD diet. Hepatic uptake transporters Oatp1a1 and Oatp1b2 mRNA levels were decreased in WT mice fed the MCD diet, whereas the efflux transporter Mrp4 was increased. However, in the H-RXR{alpha}-null mice, the MCD diet only moderately decreased Oatp1a1 and induced both Oatp1a4 and Mrp4 gene expression. Whereas the MCD diet increased serum bile acid levels and alkaline phosphatase activity in both WT and H-RXR{alpha}-null mice, serum ALT levels were induced (2.9-fold) only in the H-RXR{alpha}-null mice. In conclusion, these data suggest a critical role for RXR{alpha} in hepatic fatty acid homeostasis and protection against MCD-induced hepatocyte injury.

  13. Copper Nanoparticles and Copper Sulphate Induced Cytotoxicity in Hepatocyte Primary Cultures of Epinephelus coioides

    PubMed Central

    Wang, Tao; Chen, Xiaoyan; Long, Xiaohua; Liu, Zhaopu; Yan, Shaohua

    2016-01-01

    Copper nanoparticles (Cu-NPs) were widely used in various industrial and commercial applications. The aim of this study was to analyze the cytotoxicity of Cu-NPs on primary hepatocytes of E.coioides compared with copper sulphate (CuSO4). Cultured cells were exposed to 0 or 2.4 mg Cu L-1 as CuSO4or Cu-NPs for 24-h. Results showed either form of Cu caused a dramatic loss in cell viability, more so in the CuSO4 than Cu-NPs treatment. Compared to control, either CuSO4 or Cu-NPs significantly increased reactive oxygen species(ROS) and malondialdehyde(MDA) concentration in hepatocytes by overwhelming total superoxide dismutase (T-SOD) activity, catalase(CAT) activity and glutathione(GSH) concentration. In addition, the antioxidative-related genes [SOD (Cu/Zn), SOD (Mn), CAT, GPx4] were also down-regulated. The apoptosis and necrosis percentage was significantly higher after the CuSO4 or Cu-NPs treatment than the control. The apoptosis was induced by the increased cytochrome c concentration in the cytosol and elevated caspase-3, caspase-8 and caspase-9 activities. Additionally, the apoptosis-related genes (p53, p38β and TNF-α) and protein (p53 protein) were up-regulated after the CuSO4 or Cu-NPs treatment, with CuSO4 exposure having a greater effect than Cu-NPs. In conclusion, Cu-NPs had similar types of toxic effects as CuSO4 on primary hepatocytes of E.coioides, but toxicity of CuSO4 was more severe than that of Cu-NPs. PMID:26890000

  14. Copper Nanoparticles and Copper Sulphate Induced Cytotoxicity in Hepatocyte Primary Cultures of Epinephelus coioides.

    PubMed

    Wang, Tao; Chen, Xiaoyan; Long, Xiaohua; Liu, Zhaopu; Yan, Shaohua

    2016-01-01

    Copper nanoparticles (Cu-NPs) were widely used in various industrial and commercial applications. The aim of this study was to analyze the cytotoxicity of Cu-NPs on primary hepatocytes of E.coioides compared with copper sulphate (CuSO4). Cultured cells were exposed to 0 or 2.4 mg Cu L-1 as CuSO4or Cu-NPs for 24-h. Results showed either form of Cu caused a dramatic loss in cell viability, more so in the CuSO4 than Cu-NPs treatment. Compared to control, either CuSO4 or Cu-NPs significantly increased reactive oxygen species(ROS) and malondialdehyde(MDA) concentration in hepatocytes by overwhelming total superoxide dismutase (T-SOD) activity, catalase(CAT) activity and glutathione(GSH) concentration. In addition, the antioxidative-related genes [SOD (Cu/Zn), SOD (Mn), CAT, GPx4] were also down-regulated. The apoptosis and necrosis percentage was significantly higher after the CuSO4 or Cu-NPs treatment than the control. The apoptosis was induced by the increased cytochrome c concentration in the cytosol and elevated caspase-3, caspase-8 and caspase-9 activities. Additionally, the apoptosis-related genes (p53, p38β and TNF-α) and protein (p53 protein) were up-regulated after the CuSO4 or Cu-NPs treatment, with CuSO4 exposure having a greater effect than Cu-NPs. In conclusion, Cu-NPs had similar types of toxic effects as CuSO4 on primary hepatocytes of E.coioides, but toxicity of CuSO4 was more severe than that of Cu-NPs.

  15. Increased hepcidin in transferrin-treated thalassemic mice correlates with increased liver BMP2 expression and decreased hepatocyte ERK activation.

    PubMed

    Chen, Huiyong; Choesang, Tenzin; Li, Huihui; Sun, Shuming; Pham, Petra; Bao, Weili; Feola, Maria; Westerman, Mark; Li, Guiyuan; Follenzi, Antonia; Blanc, Lionel; Rivella, Stefano; Fleming, Robert E; Ginzburg, Yelena Z

    2016-03-01

    Iron overload results in significant morbidity and mortality in β-thalassemic patients. Insufficient hepcidin is implicated in parenchymal iron overload in β-thalassemia and approaches to increase hepcidin have therapeutic potential. We have previously shown that exogenous apo-transferrin markedly ameliorates ineffective erythropoiesis and increases hepcidin expression in Hbb(th1/th1) (thalassemic) mice. We utilize in vivo and in vitro systems to investigate effects of exogenous apo-transferrin on Smad and ERK1/2 signaling, pathways that participate in hepcidin regulation. Our results demonstrate that apo-transferrin increases hepcidin expression in vivo despite decreased circulating and parenchymal iron concentrations and unchanged liver Bmp6 mRNA expression in thalassemic mice. Hepatocytes from apo-transferrin-treated mice demonstrate decreased ERK1/2 pathway and increased serum BMP2 concentration and hepatocyte BMP2 expression. Furthermore, hepatocyte ERK1/2 phosphorylation is enhanced by neutralizing anti-BMP2/4 antibodies and suppressed in vitro in a dose-dependent manner by BMP2, resulting in converse effects on hepcidin expression, and hepatocytes treated with MEK/ERK1/2 inhibitor U0126 in combination with BMP2 exhibit an additive increase in hepcidin expression. Lastly, bone marrow erythroferrone expression is normalized in apo-transferrin treated thalassemic mice but increased in apo-transferrin injected wild-type mice. These findings suggest that increased hepcidin expression after exogenous apo-transferrin is in part independent of erythroferrone and support a model in which apo-transferrin treatment in thalassemic mice increases BMP2 expression in the liver and other organs, decreases hepatocellular ERK1/2 activation, and increases nuclear Smad to increase hepcidin expression in hepatocytes.

  16. A role for caspase-8 and c-FLIPL in proliferation and cell-cycle progression of primary hepatocytes.

    PubMed

    Gilot, David; Serandour, Anne-Laure; Ilyin, Guennady P; Lagadic-Gossmann, Dominique; Loyer, Pascal; Corlu, Anne; Coutant, Alexandre; Baffet, Georges; Peter, Marcus E; Fardel, Olivier; Guguen-Guillouzo, Christiane

    2005-12-01

    Growth factors are known to favor both proliferation and survival of hepatocytes. In the present study, we investigated if c-FLIP(L) (cellular FLICE-inhibitory protein, long isoform) could be involved in epidermal growth factor (EGF)-stimulated proliferation of rat hepatocytes since c-FLIP(L) regulates both cell proliferation and procaspase-8 maturation. Treatment with MEK inhibitors prevented induction of c-FLIP(L) by EGF along with total inhibition of DNA replication. However, EGF failed to inhibit processing of procaspase-8 in the presence of EGF suggesting that c-FLIP(L) does not play its canonical anti-apoptotic role in this model. Downregulation of c-FLIP expression using siRNA oligonucleotides strongly reduced DNA replication but did not result in enhanced apoptosis. Moreover, intermediate cleavage products of c-FLIP(L) and caspase-8 were found in EGF-treated hepatocytes in the absence of caspase-3 maturation and cell death. To determine whether the Fas/FADD/caspase-8/c-FLIP(L) complex was required for this activity, Fas, procaspase-8 and Fas-associated death domain protein (FADD) expression or function was inhibited using siRNA or constructs encoding dominant negative mutant proteins. Inhibition of any of these components of the Fas/FADD/caspase-8 pathway decreased DNA replication suggesting a function of these proteins in cell-cycle arrest. Similar results were obtained when the IETD-like caspase activity detectable in EGF-treated hepatocytes was inhibited by the pan-caspase inhibitor, z-ASP. Finally, we demonstrated co-immunoprecipitation between EGFR and Fas within 15 min following EGF stimulation. In conclusion, our results indicate that the Fas/FADD/c-FLIP(L)/caspase-8 pathway positively controls the G(1)/S transition in EGF-stimulated hepatocytes. Our data provide new insights into the mechanisms by which apoptotic proteins participate to mitogenic signals during the G(1) phase.

  17. A semi-mechanistic integrated toxicokinetic–toxicodynamic (TK/TD) model for arsenic(III) in hepatocytes

    PubMed Central

    Stamatelos, Spyros K.; Androulakis, Ioannis P.; Kong, Ah-Ng Tony; Georgopoulos, Panos G.

    2014-01-01

    Background A systems engineering approach is presented for describing the kinetics and dynamics that are elicited upon arsenic exposure of human hepatocytes. The mathematical model proposed here tracks the cellular reaction network of inorganic and organic arsenic compounds present in the hepatocyte and analyzes the production of toxicologically potent by-products and the signaling they induce in hepatocytes. Methods and results The present modeling effort integrates for the first time a cellular-level semi-mechanistic toxicokinetic (TK) model of arsenic in human hepatocytes with a cellular-level toxicodynamic (TD) model describing the arsenic-induced reactive oxygen species (ROS) burst, the antioxidant response, and the oxidative DNA damage repair process. The antioxidant response mechanism is described based on the Keap1-independent Nuclear Factor-erythroid 2-related factor 2 (Nrf2) signaling cascade and accounts for the upregulation of detoxifying enzymes. The ROS-induced DNA damage is simulated by coupling the TK/TD formulation with a model describing the multistep pathway of oxidative DNA repair. The predictions of the model are assessed against experimental data of arsenite-induced genotoxic damage to human hepatocytes; thereby capturing in silico the mode of the experimental dose–response curve. Conclusions The integrated cellular-level TK/TD model presented here provides significant insight into the underlying regulatory mechanism of Nrf2-regulated antioxidant response due to arsenic exposure. While computational simulations are in a fair good agreement with relevant experimental data, further analysis of the system unravels the role of a dynamic interplay among the feedback loops of the system in controlling the ROS upregulation and DNA damage response. This TK/TD framework that uses arsenic as an example can be further extended to other toxic or pharmaceutical agents. PMID:23069314

  18. Caffeine induces CYP1A2 expression in rat hepatocytes but not in human hepatocytes.

    PubMed

    Vaynshteyn, David; Jeong, Hyunyoung

    2012-06-01

    Caffeine is the active constituent in coffee. Continual consumption of caffeine can lead to an attenuated response also known as tolerance. Results from rat studies have shown that caffeine is an inducer of CYP1A2, the enzyme responsible for caffeine's metabolism. This suggests that CYP1A2 induction by caffeine may be in part responsible for caffeine tolerance. However, whether caffeine induces CYP1A2 expression in humans remains unknown. Our results from luciferase assays performed in HepG2 cells showed that caffeine is not an activator of the aromatic hydrocarbon receptor (AhR), a major transcription factor involved in upregulation of CYP1A2. Furthermore, caffeine did not induce CYP1A2 expression in primary human hepatocytes at a concentration attained by ordinary coffee drinking. On the other hand, caffeine enhanced CYP1A2 expression by 9-fold in rat hepatocytes. Our results suggest that caffeine from ordinary coffee drinking does not induce CYP1A2 expression in humans and that factors other than CYP1A2 induction by caffeine likely contribute to development of caffeine tolerance in humans.

  19. Differentiated properties of hepatocytes induced from pancreatic cells.

    PubMed

    Tosh, David; Shen, Chia-Ning; Slack, Jonathan M W

    2002-09-01

    Transdifferentiation of pancreas to liver is a well-recognized phenomenon and has been described in animal experiments and human pathology. We recently produced an in vitro model for the transdifferentiation (or conversion) of the pancreatic cell line AR42J-B13 to hepatocytes based on culture with dexamethasone (Dex). To determine whether the hepatocytes express markers of hepatic intermediary metabolism and detoxification, we investigated the patterns of expression of glucokinase, cytochrome P450s CYP3A1 and CYP2B1/2, testosterone/4-nitrophenol uridine diphosphate glucuronosyltransferase (UDPGT), and aryl sulfotransferase. All were expressed. We also determined the expression of 2 enzymes involved in ammonia detoxification: carbamoylphosphate synthetase I (CPS I) and glutamine synthetase (GS). These enzymes are normally strictly compartmentalized in liver in a wide periportal pattern and the last downstream perivenous hepatocytes, respectively. Following culture with Dex, CPS I and GS are expressed in 2 different cell populations, suggesting that both periportal and perivenous hepatocytes are induced. We also produced a reporter assay based on the activation of green fluorescent protein (GFP) by the transthyretin (TTR) promoter or glucose-6-phosphatase (G6Pase) promoter. After culture with Dex, transfected cells begin to express GFP, showing that hepatic promoters are activated in concert with the induction of the hepatocyte phenotype. Lastly, we examined the stability of the hepatic phenotype and found that some cells still express liver markers (transferrin or albumin) up to 14 days after removal of Dex. In conclusion, these results suggest that pancreatic hepatocytes produced by this method may offer an alternative model to primary cultures of hepatocytes for the study of liver function.

  20. Role of macrophages in the immune response to hepatocytes

    SciTech Connect

    Bumgardner, G.L.; Chen, S.; Almond, S.P.; Ascher, N.L.; Payne, W.D.; Matas, A.J. )

    1990-06-01

    The purpose of this study was to determine the role of host macrophages in the development of allospecific cytolytic T cells (allo-CTLs) in response to purified allogeneic MHC Class I+, Class II- hepatocytes in vivo in hepatocyte sponge matrix allografts (HC-SMA). Depletion of antigen-presenting cells (APCs) from responder splenocytes in mixed lymphocyte hepatocyte culture (MLHC) inhibits the development of allo-CTLs in response to purified hepatocytes. First the ability of sponge macrophages to function as accessory cells in indirect presentation of hepatocyte Class I antigen was tested in MLHC. We found that addition of irradiated sponge cells (a source of sponge macrophages) restored the development of allo-CTLs in MLHC depleted of responder APCs. Therefore, radioresistant sponge macrophages can function as accessory cells in MLHC. We next employed silica as an immunotherapy targeted against host macrophages and assessed the effect on development of allo-CTLs in HC-SMA. We found that local (intrasponge) silica treatment completely inhibited the development of allo-CTLs in HC-SMA. Combined local and systemic silica treatment resulted in inhibition of allocytotoxicity comparable to local silica treatment alone in the doses tested. We conclude that host macrophages which infiltrate HC-SMA can function as accessory cells in vitro in MLHC and that both infiltrating host macrophages and lymphocytes participate in the development of an alloimmune response to purified hepatocytes in vivo. This interaction may involve indirect antigen presentation of hepatocyte Class I antigen by macrophages to host lymphocytes which accumulate in HC-SMA.

  1. Rifampicin induction of lidocaine metabolism in cultured human hepatocytes.

    PubMed

    Li, A P; Rasmussen, A; Xu, L; Kaminski, D L

    1995-08-01

    In our laboratory, cultured human hepatocytes are being evaluated as an experimental system to study drug interactions. We report the effect of a known cytochrome P450 (CYP) inducer, rifampicin, on the metabolism of lidocaine by primary human hepatocytes. Rifampicin has been shown to induce CYP3A4, a major human hepatic CYP isozyme that is known to metabolize lidocaine to its primary metabolite, monoethylglycinexylidide. Human hepatocytes were cultured on collagen-coated plates in serum-free, hormone-supplemented Waymouth medium for a 3-day period before they were treated with rifampicin at 50 microM for 1 to 3 days. Hepatocytes isolated from five individuals were studied, and, in all cases, lidocaine metabolism was found to be induced by rifampicin, as demonstrated by a higher rate of monoethylglycinexylidide formation than concurrent controls. For three of the hepatocyte cultures, lidocaine metabolism was evaluated at various times after treatment. Induction was observed at 1 day after treatment, and reached higher levels at day 2 or 3. The level of induction was found to be approximately 100% for two hepatocyte isolations and approximately 600% for one isolation. In a separate experiment, hepatocytes were treated with rifampicin for a 2-day period. Rate of lidocaine metabolism at multiple substrate concentrations (10-120 microM) were then studied. Rifampicin induction of lidocaine metabolism (approximately 100%) was observed at all the lidocaine concentrations studied. Lineweaver-Burk plot of the data showed an increase in Vmax and a less significant change in Km. Induction of lidocaine metabolism by rifampicin (concentrations of 0.1-50 microM) was found to be dose-dependent, with significant induction observed at 1 microM and higher concentrations. (ABSTRACT TRUNCATED AT 250 WORDS)

  2. Nuclear Translocation of β-Catenin during Mesenchymal Stem Cells Differentiation into Hepatocytes Is Associated with a Tumoral Phenotype

    PubMed Central

    Herencia, Carmen; Martínez-Moreno, Julio M.; Herrera, Concepción; Corrales, Fernando; Santiago-Mora, Raquel; Espejo, Isabel; Barco, Monserrat; Almadén, Yolanda; de la Mata, Manuel; Rodríguez-Ariza, Antonio; Muñoz-Castañeda, Juan R.

    2012-01-01

    Wnt/β-catenin pathway controls biochemical processes related to cell differentiation. In committed cells the alteration of this pathway has been associated with tumors as hepatocellular carcinoma or hepatoblastoma. The present study evaluated the role of Wnt/β-catenin activation during human mesenchymal stem cells differentiation into hepatocytes. The differentiation to hepatocytes was achieved by the addition of two different conditioned media. In one of them, β-catenin nuclear translocation, up-regulation of genes related to the Wnt/β-catenin pathway, such as Lrp5 and Fzd3, as well as the oncogenes c-myc and p53 were observed. While in the other protocol there was a Wnt/β-catenin inactivation. Hepatocytes with nuclear translocation of β-catenin also had abnormal cellular proliferation, and expressed membrane proteins involved in hepatocellular carcinoma, metastatic behavior and cancer stem cells. Further, these cells had also increased auto-renewal capability as shown in spheroids formation assay. Comparison of both differentiation protocols by 2D-DIGE proteomic analysis revealed differential expression of 11 proteins with altered expression in hepatocellular carcinoma. Cathepsin B and D, adenine phosphoribosyltransferase, triosephosphate isomerase, inorganic pyrophosphatase, peptidyl-prolyl cis-trans isomerase A or lactate dehydrogenase β-chain were up-regulated only with the protocol associated with Wnt signaling activation while other proteins involved in tumor suppression, such as transgelin or tropomyosin β-chain were down-regulated in this protocol. In conclusion, our results suggest that activation of the Wnt/β-catenin pathway during human mesenchymal stem cells differentiation into hepatocytes is associated with a tumoral phenotype. PMID:22506042

  3. Experimental Model for Successful Liver Cell Therapy by Lenti TTR-YapERT2 Transduced Hepatocytes with Tamoxifen Control of Yap Subcellular Location

    PubMed Central

    Yovchev, Mladen; Jaber, Fadi L.; Lu, Zhonglei; Patel, Shachi; Locker, Joseph; Rogler, Leslie E.; Murray, John W.; Sudol, Marius; Dabeva, Mariana D.; Zhu, Liang; Shafritz, David A.

    2016-01-01

    Liver repopulation by transplanted hepatocytes has not been achieved previously in a normal liver microenvironment. Here we report that adult rat hepatocytes transduced ex vivo with a lentivirus expressing a human YapERT2 fusion protein (hYapERT2) under control of the hepatocyte-specific transthyretin (TTR) promoter repopulate normal rat liver in a tamoxifen-dependent manner. Transplanted hepatocytes expand very slowly but progressively to produce 10% repopulation at 6 months, showing clusters of mature hepatocytes that are fully integrated into hepatic parenchyma, with no evidence for dedifferentiation, dysplasia or malignant transformation. Thus, we have developed the first vector designed to regulate the growth control properties of Yap that renders it capable of producing effective cell therapy. The level of liver repopulation achieved has significant translational implications, as it is 2-3x the level required to cure many monogenic disorders of liver function that have no underlying hepatic pathology and is potentially applicable to diseases of other tissues and organs. PMID:26763940

  4. Morphology and function of isolated hepatocytes transplanted into rat spleen.

    PubMed

    Mito, M; Ebata, H; Kusano, M; Onishi, T; Saito, T; Sakamoto, S

    1979-12-01

    Hepatocytes isolated by the collagenase digestive method were transplanted into the spleens of syngeneic rats. Morphology and function of the hepatocytes in the spleen were investigated for 12 to 17 months after transplantation. The transplanted hepatocytes proliferated and reconfigured in the spleen without direct perfusion of portal venous blood and with the presence of an intact host liver. Fourteen to 17 months after transplantation, the hepatocytes which had formed a demarcated nodule occupied approximately 40% of the area of the splenic parenchyma without undifferentiation on microscopic examination. However, the weight of the hepatized spleen did not increase beyond the weight of a normal spleen and the weight of the host liver that had normal morphology also did not differ from a normal liver. Light and electron microscopic studies demonstrated differentiated cord structure and normal architecture for each heptocyte. Furthermore, the hepatized spleen synthesized albumin and glycogen as demonstrated by immunofluorescence and histochemical studies. Ammonia tolerance and indocyanine green clearance tests revealed functioning hepatocytes in the spleen proper. These results indicate that our experimental model lends itself well to investigations in cell growth mechanism and that hepatocellular transplantation has potential clinical application to compensate for impaired hepatic function.

  5. Molecular cytotoxic mechanisms of chlorpromazine in isolated rat hepatocytes.

    PubMed

    MacAllister, Stephanie L; Young, Cheryl; Guzdek, Anna; Zhidkov, Nickholas; O'Brien, Peter J

    2013-01-01

    Chlorpromazine (CPZ), a member of the largest class of first-generation antipsychotic agents, is known to cause hepatotoxicity in the form of cholestasis and hepatocellular necrosis in some patients. The mechanism of CPZ hepatotoxicity is unclear, but is thought to result from reactive metabolite formation. The goal of this research was to assess potential cytotoxic mechanisms of CPZ using the accelerated cytotoxicity mechanism screening (ACMS) technique with freshly isolated rat hepatocytes. This study identified CPZ cytotoxicity and inhibition of mitochondrial membrane potential (MMP) to be concentration-dependent. Furthermore, inhibition of cytochrome P450s (CYPs), including CYP2D1 and 1A2, delayed CPZ cytotoxicity, suggesting a role for CYP activation of CPZ to a toxic metabolite(s) in this model. Metabolism studies also demonstrated glucuronide and glutathione (GSH) requirement for CPZ detoxification in hepatocytes. Inactivating the 2-electron reduction pathway, NAD(P)H quinone oxidoreductase (NQO1), caused a significant increase in hepatocyte susceptibility to CPZ, indicating quinoneimine contribution to CPZ cytotoxicity. Nontoxic concentrations of peroxidase/H(2)O(2) (inflammatory model) increased cytotoxicity in CPZ-treated hepatocytes and caused additional mitochondrial toxicity. Inflammation further depleted GSH and increased oxidized glutathione (GSSG) levels. Results suggest activation of CPZ to reactive metabolites by 2 pathways in hepatocytes: (i) a CYP-catalyzed quinoneimine pathway, and (ii) a peroxidase-catalyzed oxidation of CPZ to CPZ radicals.

  6. Hepatitis B antigen in hepatocytes of chronic active liver disease.

    PubMed

    Kawanishi, H

    1979-04-01

    To study the morphologic interrelation of hepatocytes with the replication of hepatitis B vius (HBV) and immunocompetent cells in chronic active liver disease(CALD), organ cultures were prepared from liver biopsy specimens. Replication of hepatitis B core antigen (HBcAg) appears to occur in the nucleus of the hepatocyte in close association with intranuclear electron-dense strands and sometimes intranucleolar matrixes (likely HBcAg genomes), and cytoplasmic maturation of the HBcAg takes place in the preautolytic condition of host hepatocytes. Immunocompetent cells became progressively autolyzed in the early period of cultures. No difference in progression of hepatocyte injury in tissues from normal subjects and from hepatitis B surface antigen (HBsAg)-positive and HBsAg-negative patients with CALD may suggest that intracellular synthesis of HBV alone is not cytopathic to host hepatocytes. This model is promising for the study of HBV replication and development, and also for testing the efficacy of new antiviral agents against the virus.

  7. Strategies for short-term storage of hepatocytes for repeated clinical infusions.

    PubMed

    Jorns, Carl; Gramignoli, Roberto; Saliem, Mohammed; Zemack, Helen; Mörk, Lisa-Mari; Isaksson, Bengt; Nowak, Greg; Ericzon, Bo-Göran; Strom, Stephen; Ellis, Ewa

    2014-01-01

    Hepatocyte transplantation is an upcoming treatment for patients with metabolic liver diseases. Repeated cell infusions over 1-2 days improve clinical outcome. Isolated hepatocytes are usually cold stored in preservation solutions between repeated infusions. However, during cold storage isolated hepatocytes undergo cell death. We investigated if tissue preservation and repeated isolations are better than storage of isolated hepatocytes when cold preserving human hepatocytes. Liver tissue obtained from liver surgery or organ donors was divided into two pieces. Hepatocytes were isolated by collagenase digestion. Hepatocytes were analyzed directly after isolation (fresh) or after storage for 48 h at 4°C in University of Wisconsin solution (UW cells). Liver tissue from the same donor was stored at 4°C in UW and hepatocytes were isolated after 48 h (UW tissue cells). Hepatocyte viability and function was evaluated by trypan blue exclusion, plating efficiency, ammonia metabolism, CYP 1A1/2, 2C9, 3A7, and 3A4 activities, phase II conjugation, and apoptosis evaluation by TUNEL assay and caspase-3/7 activities. Hepatocytes stored in UW showed a significantly lower viability compared to fresh cells or hepatocytes isolated from tissue stored for 48 h (54% vs. 71% vs. 79%). Plating efficiency was significantly decreased for cells stored in UW (40%) compared to fresh and UW tissue cells (63% vs. 55%). No significant differences between UW cells and UW tissue cells could be shown for CYP activities or ammonia metabolism. Hepatocytes stored in UW showed a strong increase in TUNEL-positive cells, whereas TUNEL staining in cold-stored liver tissue and hepatocytes isolated after 48 h was unchanged. This observation was confirmed by increased caspase-3/7 activities in UW cells. Although preservation of isolated hepatocytes in UW maintains function, cold storage of liver tissue and repeated hepatocyte isolations is superior to cold storage of isolated hepatocytes in preserving

  8. Effect of adrenergic agonists and antagonists on alanine amino transferase, fructose-1:6-bisphosphatase and glucose production in hepatocytes.

    PubMed

    Begum, N A; Datta, A G

    1992-08-18

    Using rat hepatocytes we confirmed our previous results that glucagon and beta-adrenergic agonists increased the enzyme activity of alanine aminotransferase (AAT) and propranolol abolished their effects. Only the enzyme activity was measured and other parameters like quantity of the enzyme or activation due to modification were not looked for. As in perfusion experiment phenylephrine and phenoxybenzamine (alpha-agonist and alpha-antagonist respectively) also alpha-antagonist respectively) also increased the AAT activity in isolated rat hepatocytes and propranolol reversed these effects. The additive effect of glucagon and phenoxybenzamine on AAT was also persistent in hepatocyte system. Fructose-1:6-bisphosphatase (Fru-P2-ase), another key enzyme in gluconeogenic pathway, was elevated by glucagon and other beta-adrenergic agonists both in liver perfusion and isolated hepatocyte experiments and was brought back to the normal level by propranolol. In this case also only the enzyme activity was measured and no other parameters were looked for. Unlike AAT this enzyme was not stimulated by phenylephrine or phenoxybenzamine. But AAT and Fru-P2-ase activities were increased significantly by adenylate cyclase activators like fluoride or forskolin. Thus, it appears that the regulation of fru-P2-ase by glucagon is purely a b-receptor mediated process whereas AAT activation shows a mixed type of regulation where some well known alpha-agonist and antagonists are behaving as beta-agonists. Results further indicate the presence of phosphodiesterase in hepatocyte membrane which was stimulated by glucagon and brought back to the normal level by propranolol. The different adrenergic compounds stated above, not only modified the activity of the above two enzymes but also stimulated glucose production by hepatocytes from alanine which was in turn abolished by propranolol as well as amino oxyacetate (AOA), a highly specified inhibitor of AAT. This confirm the participation of AAT in

  9. Cell Fusion Reprogramming Leads to a Specific Hepatic Expression Pattern during Mouse Bone Marrow Derived Hepatocyte Formation In Vivo

    PubMed Central

    Arza, Elvira; Alvarez-Barrientos, Alberto; Fabregat, Isabel; Garcia-Bravo, Maria; Meza, Nestor W.; Segovia, Jose C.

    2012-01-01

    The fusion of bone marrow (BM) hematopoietic cells with hepatocytes to generate BM derived hepatocytes (BMDH) is a natural process, which is enhanced in damaged tissues. However, the reprogramming needed to generate BMDH and the identity of the resultant cells is essentially unknown. In a mouse model of chronic liver damage, here we identify a modification in the chromatin structure of the hematopoietic nucleus during BMDH formation, accompanied by the loss of the key hematopoietic transcription factor PU.1/Sfpi1 (SFFV proviral integration 1) and gain of the key hepatic transcriptional regulator HNF-1A homeobox A (HNF-1A/Hnf1a). Through genome-wide expression analysis of laser captured BMDH, a differential gene expression pattern was detected and the chromatin changes observed were confirmed at the level of chromatin regulator genes. Similarly, Tranforming Growth Factor-β1 (TGF-β1) and neurotransmitter (e.g. Prostaglandin E Receptor 4 [Ptger4]) pathway genes were over-expressed. In summary, in vivo BMDH generation is a process in which the hematopoietic cell nucleus changes its identity and acquires hepatic features. These BMDHs have their own cell identity characterized by an expression pattern different from hematopoietic cells or hepatocytes. The role of these BMDHs in the liver requires further investigation. PMID:22457803

  10. Production of hepatocyte like cells from human pluripotent stem cells

    PubMed Central

    Hannan, Nicholas R.F; Segeritz, Charis-Patricia; Touboul, Thomas; Vallier, Ludovic

    2013-01-01

    Large scale production of hepatocytes from a variety of genetic backgrounds would be beneficial for drug screening and to provide a source of cells to be used as a substitute for liver transplantation. However, fully functional primary hepatocytes remain difficult to expand in vitro and circumventing this problem by using an alternative source of cells is desirable. Here, we describe a 25 day protocol to direct the differentiation of human pluripotent stem cells into a near homogenous population of hepatocyte-like cells. As cells progress through this protocol they express genes in a chronological manner similar to that described during in-vivo hepatic development. The protocol relies on culture systems devoid of serum, feeders or complex extra-cellular matrices enabling molecular analyses without interference from unknown factors. This approach works efficiently with human embryonic stem cells and human induced pluripotent stem cells and was recently used to model liver diseases in vitro. PMID:23424751

  11. Alternative Cell Sources to Adult Hepatocytes for Hepatic Cell Therapy.

    PubMed

    Pareja, Eugenia; Gómez-Lechón, María José; Tolosa, Laia

    2017-01-01

    Adult hepatocyte transplantation is limited by scarce availability of suitable donor liver tissue for hepatocyte isolation. New cell-based therapies are being developed to supplement whole-organ liver transplantation, to reduce the waiting-list mortality rate, and to obtain more sustained and significant metabolic correction. Fetal livers and unsuitable neonatal livers for organ transplantation have been proposed as potential useful sources of hepatic cells for cell therapy. However, the major challenge is to use alternative cell sources for transplantation that can be derived from reproducible methods. Different types of stem cells with hepatic differentiation potential are eligible for generating large numbers of functional hepatocytes for liver cell therapy to treat degenerative disorders, inborn hepatic metabolic diseases, and organ failure. Clinical trials are designed to fully establish the safety profile of such therapies and to define target patient groups and standardized protocols.

  12. Determination of metabolic stability using cryopreserved hepatocytes from rainbow trout (Oncorhynchus mykiss)

    EPA Science Inventory

    Standard protocols for isolating, cryopreserving, and thawing rainbow trout hepatocytes are described, along with procedures for using fresh or cryopreserved hepatocytes to assess chemical metabolic stability in fish by means of a substrate depletion approach. Variations on thes...

  13. Cryopreservation of isolated human hepatocytes for transplantation: State of the art.

    PubMed

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

    2006-10-01

    Hepatocytes isolated from unused donor livers are being used for transplantation in patients with acute liver failure and liver-based metabolic defects. As large numbers of hepatocytes can be prepared from a single liver and hepatocytes need to be available for emergency and repeated treatment of patients it is essential to be able to cryopreserve and store cells with good thawed cell function. This review considers the current status of cryopreservation of human hepatocytes discussing the different stages involved in the process. These include pre-treatment of cells, freezing solution, cryoprotectants and freezing and thawing protocols. There are detrimental effects of cryopreservation on hepatocyte structure and metabolic function, including cell attachment, which is important to the engraftment of transplanted cells in the liver. Cryopreserved human hepatocytes have been successfully used in clinical transplantation, with evidence of replacement of missing function. Further optimisation of hepatocyte cryopreservation protocols is important for their use in hepatocyte transplantation.

  14. Hepatitis C virus represses E-cadherin expression via DNA methylation to induce epithelial to mesenchymal transition in human hepatocytes.

    PubMed

    Park, Jungmi; Jang, Kyung Lib

    2014-04-04

    Hepatitis C virus (HCV) core protein is known to induce promoter hypermethylation of tumor suppressor genes including E-cadherin to repress their expression when overexpressed in human hepatocytes; however, its actual role during HCV infection is still unknown. Here, we report that infection with HCV derived from pJFH-1 replicon system that mimics natural infection elevates protein levels of DNA methyltransferase 1 and 3b to enhance DNMT activity in human hepatocytes. As a consequence, HCV induced promoter hypermethylation of E-cadherin, resulting in repression of its expression. In addition down-regulation of E-cadherin by HCV led to epithelial-mesenchymal transition that is known to be a critical event during the late stage of tumorigenesis.

  15. Transport in isolated rat hepatocytes of the phospho-oligosaccharide that mimics insulin action. Effects of adrenalectomy and glucocorticoid treatment.

    PubMed Central

    Alvarez, J F; Sánchez-Arias, J A; Guadaño, A; Estévez, F; Varela, I; Felíu, J E; Mato, J M

    1991-01-01

    The addition to intact cells of an inositol phospho-oligosaccharide (POS), which is the polar head-group of an insulin-sensitive glycosylphosphatidylinositol, mimics and may mediate some of the biological effects of this hormone. Here we report the existence of a POS transport system in hepatocytes. This POS transport system is specific and time- and dose-dependent. Insulin-resistance caused by dexamethasone administration to rats was accompanied by a decrease in the hepatocyte POS transport system. In contrast, bilateral adrenalectomy provoked a significant increase in the transport of POS. Both the temporal uptake of POS and the regulation of this process by conditions known to modify the sensitivity to insulin suggest that this novel transport system might be involved in the insulin signalling mechanism. PMID:2006906

  16. In vivo hepatocyte MR imaging using lactose functionalized magnetoliposomes.

    PubMed

    Ketkar-Atre, Ashwini; Struys, Tom; Dresselaers, Tom; Hodenius, Michael; Mannaerts, Inge; Ni, Yicheng; Lambrichts, Ivo; Van Grunsven, Leo A; De Cuyper, Marcel; Himmelreich, Uwe

    2014-01-01

    The aim of this study was to assess a novel lactose functionalized magnetoliposomes (MLs) as an MR contrast agent to target hepatocytes as well as to evaluate the targeting ability of MLs for in vivo applications. In the present work, 17 nm sized iron oxide cores functionalized with anionic MLs bearing lactose moieties were used for targeting the asialoglycoprotein receptor (ASGP-r), which is highly expressed in hepatocytes. Non-functionalized anionic MLs were tested as negative controls. The size distribution of lactose and anionic MLs was determined by transmission electron microscopy (TEM) and dynamic light scattering (DLS). After intravenous administration of both MLs, contrast enhancement in the liver was observed by magnetic resonance imaging (MRI). Label retention was monitored non-invasively by MRI and validated with Prussian blue staining and TEM for up to eight days post MLs administration. Although the MRI signal intensity did not show significant differences between functionalized and non-functionalized particles, iron-specific Prussian blue staining and TEM analysis confirmed the uptake of lactose MLs mainly in hepatocytes. In contrast, non-functionalized anionic MLs were mainly taken up by Kupffer and sinusoidal cells. Target specificity was further confirmed by high-resolution MR imaging of phantoms containing isolated hepatocytes, Kupffer cell (KCs) and hepatic stellate cells (HSCs) fractions. Hypointense signal was observed for hepatocytes isolated from animals which received lactose MLs but not from animals which received anionic MLs. These data demonstrate that galactose-functionalized MLs can be used as a hepatocyte targeting MR contrast agent to potentially aid in the diagnosis of hepatic diseases if the non-specific uptake by KCs is taken into account.

  17. Intrasplenic transplantation of allogeneic hepatocytes prolongs survival in anhepatic rats.

    PubMed

    Arkadopoulos, N; Lilja, H; Suh, K S; Demetriou, A A; Rozga, J

    1998-11-01

    To examine whether hepatocytes transplanted in the spleen can function as an ectopic liver, we performed hepatocyte transplantation in rats that were rendered anhepatic. Total hepatectomy was performed by using a novel single-stage technique. Following hepatectomy, Group 1 rats (n = 16) were monitored until death to determine survival time without prior intervention. Group 2 anhepatic rats (n = 20) were sacrificed at various times to measure blood hepatocyte growth factor (HGF) and transforming growth factor beta1 (TGF-beta1) levels. Group 3 (n = 16) rats received intrasplenic injection of isolated hepatocytes (2.5 x 10(7) cells/rat) followed by total hepatectomy after 3 days. Group 4 (n = 12) sham-transplanted rats received intrasplenic saline infusion, and after 3 days they were rendered anhepatic. Group 2, 3, and 4 rats were maintained on daily Cyclosporine A (10 mg/kg; intramuscularly). Group 1 anhepatic rats survived for 22.4 +/- 5.2 hours (standard deviation). The anhepatic state was associated with a progressive and statistically significant rise in blood HGF and TGF-beta1 levels. Rats that received hepatocyte transplantation before total hepatectomy had a significantly longer survival time than sham-transplanted anhepatic controls (34.1 +/- 8.5 vs. 15.5 +/- 4.8 hrs, P < .01). Additionally, at 12 hours post-hepatectomy, transplanted rats had significantly lower blood ammonia, prothrombin time, international normalized ratio, and TGF-beta1 levels when compared with sham-transplanted controls. In conclusion, intrasplenic transplantation of allogeneic hepatocytes prolonged survival, improved blood chemistry, and lowered blood TGF-beta1 levels in rats rendered anhepatic.

  18. Lipid-mediated transfection of normal adult human hepatocytes in primary culture.

    PubMed

    Ourlin, J C; Vilarem, M J; Daujat, M; Harricane, M C; Domergue, J; Joyeux, H; Baulieux, J; Maurel, P

    1997-04-05

    The aim of this work was to develop a procedure for the lipid-mediated transfection of DNA into normal adult human hepatocytes in culture. Cells were plated in a serum-free culture medium at various cell densities, on plastic or collagen-coated dishes, both in the absence and in the presence of epidermal growth factor (EGF). The cells were incubated for various periods of time with mixtures of DNA-lipofectin or DNA-3 beta[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-chol) liposomes, and the efficiency of transfection was assessed by measuring the activity of reporter genes, beta-galactosidase or chloramphenicol acetyl-transferase (CAT). For comparison, similar experiments were carried out with human cell lines including HepG2, Caco-2, and WRL68. The efficiency of transfection (in percentage of cells) was not significantly different after transfection with lipofectin or DC-chol and comprised between 0.04 and 1.7% (extreme values) for different cultures. The efficiency of transfection decreased as the age or density of the culture increased and increased in cultures treated with EGF. Direct measurement of the rate of DNA synthesis suggested that the efficiency of transfection was related to the number of cells entering the S phase. Under the same conditions, the efficiency of transfection was one to two orders of magnitude greater in the three cell lines. A plasmid harboring 660 bp of the 5'-flanking region of CYP1A1 (containing two xenobiotic enhancer elements) fused upstream of the promoter of thymidine kinase and the CAT reporter gene was constructed. When this plasmid was transfected in human hepatocytes, CAT activity was induced as expected. We conclude that normal adult human hepatocytes can be transfected with exogenous DNA and that the transfected construct is regulated in the manner expected from in vivo studies.

  19. Low concentration of mercury induces autophagic cell death in rat hepatocytes.

    PubMed

    Chatterjee, Sarmishtha; Ray, Atish; Mukherjee, Sandip; Agarwal, Soumik; Kundu, Rakesh; Bhattacharya, Shelley

    2014-08-01

    In the present study, we attempted to elucidate the induction of autophagy in rat hepatocytes by a low concentration of mercury. Hepatocytes treated with different doses of mercuric chloride (HgCl2; 1, 2.5, 5 and 10 µM) and at different time intervals (0 min, 30 min, 1 h, 2 h and 4 h) show autophagic cell death only at 5 µM HgCl2 within 30 min of incubation. At 1 and 2.5 µM HgCl2, no cell death is recorded, while apoptosis is found at 10 µM HgCl2, as evidenced by the activation of caspase 3. Autophagic cell death is confirmed by the presence of monodansylcadaverine (MDC) positive hepatocytes which is found to be highest at 1 h. Atg5-Atg12 covalent-conjugation triggers the autophagic pathway within 30 min of 5 µM HgCl2 treatment and continues till 4 h of incubation. In addition, damage-regulated autophagy modulator (DRAM) expression gradually increases from 30 min to 4 h of treatment with mercury and a corresponding linear decrease in p53 has been observed. It is concluded that a low concentration (5 µM HgCl2) of mercury induces autophagy or nonapoptotic programmed cell death following an Atg5-Atg12 covalent-conjugation pathway, which is modulated by DRAM in a p53-dependent manner.

  20. New role of irisin in hepatocytes: The protective effect of hepatic steatosis in vitro.

    PubMed

    Park, Min-Jung; Kim, Dong-Il; Choi, Joo-Hee; Heo, Young-Ran; Park, Soo-Hyun

    2015-09-01

    Irisin is a newly identified myokine related to exercise and the browning of white fat. Recently, it was reported that irisin serum levels are associated with intrahepatic triglyceride content, suggesting that it might have an important role in the liver. The aim of this study was to determine the role of irisin in hepatocytes. Specifically, the effect of recombinant irisin on palmitic acid (PA)-induced lipogenesis and its related signal pathways were examined in AML12 cells and mouse primary hepatocytes. In the present study, we observed the presence of irisin inside the cells in response to the treatment of recombinant irisin by flow cytometry and cell imaging technique. Recombinant irisin significantly inhibited the PA-induced increase in lipogenic markers ACC and FAS at the mRNA and protein levels, and prevented the PA-induced lipid accumulation in hepatocytes. Additionally, irisin inhibited the PA-induced increase in the expression, nuclear localization, and transcriptional activities of the master regulators of lipogenesis (LXRα and SREBP-1c). Moreover, irisin attenuated PA-induced oxidative stress, which was confirmed by measuring the expression of inflammatory markers (NFκB, COX-2, p38 MAPK, TNF, IL-6) and superoxide indicator (dihydroethidium). The preventive effects of irisin against lipogenesis and oxidative stress were mediated by the inhibition of protein arginine methyltransferase-3 (PRMT3). These findings suggested that irisin might have a beneficial role in the prevention of hepatic steatosis by altering the expression of lipogenic genes and attenuating oxidative stress in a PRMT3 dependent manner.

  1. Perinatal deiodinase 2 expression in hepatocytes defines epigenetic susceptibility to liver steatosis and obesity

    PubMed Central

    Fonseca, Tatiana L.; Fernandes, Gustavo W.; McAninch, Elizabeth A.; Bocco, Barbara M. L. C.; Abdalla, Sherine M.; Ribeiro, Miriam O.; Mohácsik, Petra; Fekete, Csaba; Li, Daofeng; Xing, Xiaoyun; Wang, Ting; Gereben, Balázs; Bianco, Antonio C.

    2015-01-01

    Thyroid hormone binds to nuclear receptors and regulates gene transcription. Here we report that in mice, at around the first day of life, there is a transient surge in hepatocyte type 2 deiodinase (D2) that activates the prohormone thyroxine to the active hormone triiodothyronine, modifying the expression of ∼165 genes involved in broad aspects of hepatocyte function, including lipid metabolism. Hepatocyte-specific D2 inactivation (ALB-D2KO) is followed by a delay in neonatal expression of key lipid-related genes and a persistent reduction in peroxisome proliferator-activated receptor-γ expression. Notably, the absence of a neonatal D2 peak significantly modifies the baseline and long-term hepatic transcriptional response to a high-fat diet (HFD). Overall, changes in the expression of approximately 400 genes represent the HFD response in control animals toward the synthesis of fatty acids and triglycerides, whereas in ALB-D2KO animals, the response is limited to a very different set of only approximately 200 genes associated with reverse cholesterol transport and lipase activity. A whole genome methylation profile coupled to multiple analytical platforms indicate that 10–20% of these differences can be related to the presence of differentially methylated local regions mapped to sites of active/suppressed chromatin, thus qualifying as epigenetic modifications occurring as a result of neonatal D2 inactivation. The resulting phenotype of the adult ALB-D2KO mouse is dramatic, with greatly reduced susceptibility to diet-induced steatosis, hypertriglyceridemia, and obesity. PMID:26508642

  2. Hepatocyte specific expression of an oncogenic variant of β-catenin results in cholestatic liver disease

    PubMed Central

    Lemberger, Ursula J.; Fuchs, Claudia D.; Karer, Matthias; Haas, Stefanie; Stojakovic, Tatjana; Schöfer, Christian; Marschall, Hanns-Ulrich; Wrba, Fritz; Taketo, Makoto M.; Egger, Gerda; Trauner, Michael; Österreiche, Christophr H.

    2016-01-01

    Background The Wnt/β-catenin signaling pathway plays a crucial role in embryonic development, tissue homeostasis, wound healing and malignant transformation in different organs including the liver. The consequences of continuous β-catenin signaling in hepatocytes remain elusive. Results Livers of Ctnnb1CA hep mice were characterized by disturbed liver architecture, proliferating cholangiocytes and biliary type of fibrosis. Serum ALT and bile acid levels were significantly increased in Ctnnb1CA hep mice. The primary bile acid synthesis enzyme Cyp7a1 was increased whereas Cyp27 and Cyp8b1 were reduced in Ctnnb1CA hep mice. Expression of compensatory bile acid transporters including Abcb1, Abcb4, Abcc2 and Abcc4 were significantly increased in Ctnnb1CA hep mice while Ntcp was reduced. Accompanying changes of bile acid transporters favoring excretion of bile acids were observed in intestine and kidneys of Ctnnb1CA hep mice. Additionally, disturbed bile acid regulation through the FXR-FGF15-FGFR4 pathway was observed in mice with activated β-catenin. Materials and Methods Mice with a loxP-flanked exon 3 of the Ctnnb1 gene were crossed to Albumin-Cre mice to obtain mice with hepatocyte-specific expression of a dominant stable form of β-catenin (Ctnnb1CA hep mice). Ctnnb1CA hep mice were analyzed by histology, serum biochemistry and mRNA profiling. Conclusions Expression of a dominant stable form of β-catenin in hepatocytes results in severe cholestasis and biliary type fibrosis. PMID:27895309

  3. Evidence for two Ca2(+)-mobilizing purinoceptors on rat hepatocytes.

    PubMed Central

    Dixon, C J; Woods, N M; Cuthbertson, K S; Cobbold, P H

    1990-01-01

    Aequorin measurements of cytosolic free Ca2+ in single rat hepatocytes show that ADP and ATP, thought to act through the same P2Y purinoceptor, elicited very different responses in the majority of cells tested. ADP invariably induced transients of short duration (approx. 9 s), whereas ATP induced either similar transients or transients with a much longer duration (approx. 49 s). We explain this variability in terms of two separate purinoceptors on rat hepatocytes, one of which responds to either ATP or ADP to generate free-Ca2+ transients of short duration, and the other responds to ATP only, with transients of longer duration. PMID:2386488

  4. Interspecies differences in metabolism of arsenic by cultured primary hepatocytes

    SciTech Connect

    Drobna, Zuzana; Walton, Felecia S.; Harmon, Anne W.; Thomas, David J.; Styblo, Miroslav

    2010-05-15

    Biomethylation is the major pathway for the metabolism of inorganic arsenic (iAs) in many mammalian species, including the human. However, significant interspecies differences have been reported in the rate of in vivo metabolism of iAs and in yields of iAs metabolites found in urine. Liver is considered the primary site for the methylation of iAs and arsenic (+3 oxidation state) methyltransferase (As3mt) is the key enzyme in this pathway. Thus, the As3mt-catalyzed methylation of iAs in the liver determines in part the rate and the pattern of iAs metabolism in various species. We examined kinetics and concentration-response patterns for iAs methylation by cultured primary hepatocytes derived from human, rat, mice, dog, rabbit, and rhesus monkey. Hepatocytes were exposed to [{sup 73}As]arsenite (iAs{sup III}; 0.3, 0.9, 3.0, 9.0 or 30 nmol As/mg protein) for 24 h and radiolabeled metabolites were analyzed in cells and culture media. Hepatocytes from all six species methylated iAs{sup III} to methylarsenic (MAs) and dimethylarsenic (DMAs). Notably, dog, rat and monkey hepatocytes were considerably more efficient methylators of iAs{sup III} than mouse, rabbit or human hepatocytes. The low efficiency of mouse, rabbit and human hepatocytes to methylate iAs{sup III} was associated with inhibition of DMAs production by moderate concentrations of iAs{sup III} and with retention of iAs and MAs in cells. No significant correlations were found between the rate of iAs methylation and the thioredoxin reductase activity or glutathione concentration, two factors that modulate the activity of recombinant As3mt. No associations between the rates of iAs methylation and As3mt protein structures were found for the six species examined. Immunoblot analyses indicate that the superior arsenic methylation capacities of dog, rat and monkey hepatocytes examined in this study may be associated with a higher As3mt expression. However, factors other than As3mt expression may also contribute to

  5. [Current status and future perspectives of hepatocyte transplantation].

    PubMed

    Pareja, Eugenia; Cortés, Miriam; Gómez-Lechón, M José; Maupoey, Javier; San Juan, Fernando; López, Rafael; Mir, Jose

    2014-02-01

    The imbalance between the number of potential beneficiaries and available organs, originates the search for new therapeutic alternatives, such as Hepatocyte transplantation (HT).Even though this is a treatment option for these patients, the lack of unanimity of criteria regarding indications and technique, different cryopreservation protocols, as well as the different methodology to assess the response to this therapy, highlights the need of a Consensus Conference to standardize criteria and consider future strategies to improve the technique and optimize the results.Our aim is to review and update the current state of hepatocyte transplantation, emphasizing the future research attempting to solve the problems and improve the results of this treatment.

  6. Coculture with mesenchymal stem cells results in improved viability and function of human hepatocytes.

    PubMed

    Fitzpatrick, Emer; Wu, Yue; Dhadda, Paramjeet; Hughes, Robin D; Mitry, Ragai R; Qin, Hong; Lehec, Sharon C; Heaton, Nigel D; Dhawan, Anil

    2015-01-01

    Hepatocyte transplantation is becoming an accepted therapy for acute liver failure, either as a bridge to liver regeneration or to organ transplantation. Hepatocytes provide liver function in place of the failing organ. The maintenance of sufficient viability and function of the transplanted hepatocytes is a concern. There is a lot of recent interest in mesenchymal stem cells (MSCs) for the provision of structural and trophic support to hepatocytes, but few studies currently use primary human hepatocytes. The aim of this study was to investigate if coculture of human MSCs with cryopreserved human hepatocytes may improve their function and viability, thus with potential for cellular therapy of liver disease. MSCs were isolated from human umbilical cord or adipose tissue. Hepatocytes were isolated from donor organs unsuitable for transplantation. MSCs and hepatocytes were cocultured in both direct and indirect contact. Conditioned medium (CM) from cocultured MSCs and hepatocytes was also used on hepatocytes. Viability and liver-specific function were compared between test and controls. Human hepatocytes that were cocultured directly with MSCs demonstrated improved production of albumin from day 5 to day 25 of culture. This effect was most prominent at day 15. Likewise, urea production was improved in coculture from day 5 to 25. Indirect coculture demonstrated improved albumin production by day 4 (1,107 ng/ml) versus hepatocyte monoculture (940 ng/ml). Hepatocytes in CM demonstrated a nonsignificant improvement in function. The viability of cocultured hepatocytes was superior to that of monocultured cells with up to a 16% improvement. Thus, coculture of human hepatocytes with MSCs demonstrates both improved function and viability. The effect is seen mainly with direct coculture but can also be seen in indirect culture and with CM. Such coculture conditions may convey major advantages in hepatocyte survival and function for cell transplantation.

  7. Comparison of primary human hepatocytes and hepatoma cell line Hepg2 with regard to their biotransformation properties.

    PubMed

    Wilkening, Stefan; Stahl, Frank; Bader, Augustinus

    2003-08-01

    Cultures of primary hepatocytes and hepatoma cell line HepG2 are frequently used in in vitro models for human biotransformation studies. In this study, we characterized and compared the capacity of these model systems to indicate the presence of different classes of promutagens. Genotoxic sensitivity, enzyme activity, and gene expression were monitored in response to treatment with food promutagens benzo[a]pyrene, dimethylnitrosamine (DMN), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). DNA damage could be detected reliably with the comet assay in primary human hepatocytes, which were maintained in sandwich culture. All three promutagens caused DNA damage in primary cells, but in HepG2 no genotoxic effects of DMN and PhIP could be detected. We supposed that the lack of specific enzymes accounts for their inability to process these promutagens. Therefore, we quantified the expression of a broad range of genes coding for drug-metabolizing enzymes with real-time reverse transcription-polymerase chain reaction. The genes code for cytochromes p450 and, in addition, for a series of important phase II enzymes. The expression level of these genes in human hepatocytes was similar to those previously reported for human liver samples. On the other hand, expression levels in HepG2 differed significantly from that in human. Activity and expression, especially of phase I enzymes, were demonstrated to be extremely low in HepG2 cells. Up-regulation of specific genes by test substances was similar in both cell types. In conclusion, human hepatocytes are the preferred model for biotransformation in human liver, whereas HepG2 cells may be useful to study regulation of drug-metabolizing enzymes.

  8. Metabolic Consequences of TGFβ Stimulation in Cultured Primary Mouse Hepatocytes Screened from Transcript Data with ModeScore

    PubMed Central

    Hoppe, Andreas; Ilkavets, Iryna; Dooley, Steven; Holzhütter, Hermann-Georg

    2012-01-01

    TGFβ signaling plays a major role in the reorganization of liver tissue upon injury and is an important driver of chronic liver disease. This is achieved by a deep impact on a cohort of cellular functions. To comprehensively assess the full range of affected metabolic functions, transcript changes of cultured mouse hepatocytes were analyzed with a novel method (ModeScore), which predicts the activity of metabolic functions by scoring transcript expression changes with 987 reference flux distributions, which yielded the following hypotheses. TGFβ multiplies down-regulation of most metabolic functions occurring in culture stressed controls. This is especially pronounced for tyrosine degradation, urea synthesis, glucuronization capacity, and cholesterol synthesis. Ethanol degradation and creatine synthesis are down-regulated only in TGFβ treated hepatocytes, but not in the control. Among the few TGFβ dependently up-regulated functions, synthesis of various collagens is most pronounced. Further interesting findings include: down-regulation of glucose export is postponed by TGFβ, TGFβ up-regulates the synthesis capacity of ketone bodies only as an early response, TGFβ suppresses the strong up-regulation of Vanin, and TGFβ induces re-formation of ceramides and sphingomyelin. PMID:24957771

  9. Fate tracing of mature hepatocytes in mouse liver homeostasis and regeneration

    PubMed Central

    Malato, Yann; Naqvi, Syed; Schürmann, Nina; Ng, Raymond; Wang, Bruce; Zape, Joan; Kay, Mark A.; Grimm, Dirk; Willenbring, Holger

    2011-01-01

    Recent evidence has contradicted the prevailing view that homeostasis and regeneration of the adult liver are mediated by self duplication of lineage-restricted hepatocytes and biliary epithelial cells. These new data suggest that liver progenitor cells do not function solely as a backup system in chronic liver injury; rather, they also produce hepatocytes after acute injury and are in fact the main source of new hepatocytes during normal hepatocyte turnover. In addition, other evidence suggests that hepatocytes are capable of lineage conversion, acting as precursors of biliary epithelial cells during biliary injury. To test these concepts, we generated a hepatocyte fate-tracing model based on timed and specific Cre recombinase expression and marker gene activation in all hepatocytes of adult Rosa26 reporter mice with an adenoassociated viral vector. We found that newly formed hepatocytes derived from preexisting hepatocytes in the normal liver and that liver progenitor cells contributed minimally to acute hepatocyte regeneration. Further, we found no evidence that biliary injury induced conversion of hepatocytes into biliary epithelial cells. These results therefore restore the previously prevailing paradigms of liver homeostasis and regeneration. In addition, our new vector system will be a valuable tool for timed, efficient, and specific loop out of floxed sequences in hepatocytes. PMID:22105172

  10. trans-Dichloridobis[dicyclo­hex­yl(2,4,6-trimethyl­phen­yl)phosphane-κP]palladium(II)

    PubMed Central

    Buthelezi, Isaac; Chiririwa, Haleden; Ogutu, Hezron; Meijboom, Reinout

    2012-01-01

    The title compound, [PdCl2(C21H33P)2], forms a monomeric complex with a trans-square-planar coordination geometry about the PdII atom which lies on an inversion centre. The Pd—P bond lengths are 2.3760 (13) Å, while the Pd—Cl bond lengths are 2.3172 (14) Å. The observed structure was found to be closely related to that of trans-dichloridobis[dicyclo­hex­yl(phen­yl)phosphane-κP]palladium(II), [PdCl2{P(C6H11)2(C6H5)}2] [Burgoyne et al. (2012 ▶). Acta Cryst. E68, m404]. PMID:23284323

  11. New version of hex-ecs, the B-spline implementation of exterior complex scaling method for solution of electron-hydrogen scattering

    NASA Astrophysics Data System (ADS)

    Benda, Jakub; Houfek, Karel

    2016-07-01

    We provide an updated version of the program hex-ecs originally presented in Comput. Phys. Commun. 185 (2014) 2903-2912. The original version used an iterative method preconditioned by the incomplete LU factorization (ILU), which-though very stable and predictable-requires a large amount of working memory. In the new version we implemented a "separated electrons" (or "Kronecker product approximation", KPA) preconditioner as suggested by Bar-On et al., Appl. Num. Math. 33 (2000) 95-104. This preconditioner has much lower memory requirements, though in return it requires more iterations to reach converged results. By careful choice between ILU and KPA preconditioners one is able to extend the computational feasibility to larger calculations. Secondly, we added the option to run the KPA preconditioner on an OpenCL device (e.g. GPU). GPUs have generally better memory access times, which speeds up particularly the sparse matrix multiplication.

  12. trans-Dichloridobis{dicyclo-hex-yl[4-(dimethyl-amino)-phen-yl]phosphane-κP}platinum(II) dichloro-methane disolvate.

    PubMed

    Davis, Wade L; Meijboom, Reinout

    2012-12-01

    In the title complex, trans-[PtCl2{P(C6H11)2(4-Me2NC6H4)}2]·2CH2Cl2, the Pt(II) atom is located on an inversion centre, resulting in a trans-square-planar geometry. Important geometric parameters are the Pt-P and Pt-Cl bond lengths of 2.3258 (6) and 2.3106 (6) Å, respectively, and the P-Pt-Cl angles of 89.64 (2) and 90.36 (2)°. The effective cone angle for the dicyclo-hex-yl[4-(dimethyl-amino)-phen-yl]phosphane unit was calculated to be 164°. The compound crystallizes with two dichloro-methane solvent mol-ecules; one of which is severely disordered and was treated using the SQUEEZE routine in PLATON [Spek (2009 ▶). Acta Cryst. D65, 148-155].

  13. Comprehensive pharmacokinetic studies and oral bioavailability of two Mn porphyrin-based SOD mimics, MnTE-2-PyP5+ and MnTnHex-2-PyP5+

    PubMed Central

    Weitner, Tin; Kos, Ivan; Sheng, Huaxin; Tovmasyan, Artak; Reboucas, Julio S.; Fan, Ping; Warner, David S.; Vujaskovic, Zeljko; Batinic-Haberle, Ines; Spasojevic, Ivan

    2013-01-01

    The cationic, ortho Mn(III) N-alkylpyridylporphyrins (alkyl=ethyl, E, and n-hexyl, nHex) MnTE-2-PyP5+ (AEOL10113, FBC-007) and MnTnHex-2-PyP5+ have proven efficacious in numerous in vivo animal models of diseases having oxidative stress in common. The remarkable therapeutic efficacy observed is due to their: (1) ability to catalytically remove O2•− and ONOO− and other reactive species; (2) ability to modulate redox-based signaling pathways; (3) accumulation within critical cellular compartments, i.e., mitochondria; and (4) ability to cross the blood–brain barrier. The similar redox activities of both compounds are related to the similar electronic and electrostatic environments around the metal active sites, whereas their different bioavailabilities are presumably influenced by the differences in lipophilicity, bulkiness, and shape. Both porphyrins are water soluble, but MnTnHex-2-PyP5+ is approximately 4 orders of magnitude more lipophilic than MnTE-2-PyP5+, which should positively affect its ability to pass through biological membranes, making it more efficacious in vivo at lower doses. To gain insight into the in vivo tissue distribution of Mn porphyrins and its impact upon their therapeutic efficacy and mechanistic aspects of action, as well as to provide data that would ensure proper dosing regimens, we conducted comprehensive pharmacokinetic (PK) studies for 24 h after single-dose drug administration. The porphyrins were administered intravenously (iv), intraperitoneally (ip), and via oral gavage at the following doses: 10 mg/kg MnTE-2-PyP5+ and 0.5 or 2 mg/kg MnTnHex-2-PyP5+. Drug levels in plasma and various organs (liver, kidney, spleen, heart, lung, brain) were determined and PK parameters calculated (Cmax, C24 h, tmax, and AUC). Regardless of high water solubility and pentacationic charge of these Mn porphyrins, they are orally available. The oral availability (based on plasma AUCoral/AUCiv) is 23% for MnTE-2-PyP5+ and 21% for MnTnHex-2-PyP5

  14. A G {r_arrow} A transition at position IVS-11 +1 of the HEX A {alpha}-chain gene in a non-Ashkenazic Mexican Tay-Sachs infant

    SciTech Connect

    Miranda, S.R.P.; Gwon, S.; DeGasperi, R.

    1994-09-01

    Tay-Sachs disease (TSD) is an autosomal recessive storage disorder caused by a deficiency of the lysosomal enzyme, {beta}-N-acetylhexosaminidase A (Hex A), a heteropolymer composed of two polypeptides, {alpha} and {beta}. Mutations in the {alpha}-chain gene render the enzyme defective, resulting in the accumulation of g{sub m2} ganglioside in the nervous system. Deficiency of Hex A was detected in a non-Ashkenazic girl of Mexican origin indicating infantile onset of TSD. Molecular investigation of the {alpha}-chain gene excluded the typical Ashkenazic 4 bp insertion in the exon 11 and the intron 12 splice-junction mutations by Hae III and Dde I restriction analysis, respectively. Single strand conformation polymorphism (SSCP) analysis showed a different pattern in the sample where exon 11 and flanking regions were amplified in the patient DNA as compared to the migration of control DNA. Sequencing of PCR amplified genomic DNA containing exon 11 and flanking intronic regions showed a single base substitution (G {r_arrow} A) at position IVS-11 +1. This mutation creates a recognition site for the restriction enzyme Mbo II. Digestion of exon 11 and flanking regions with Mbo II demonstrated homozygosity of the patient for this mutation and heterozygosity in the mother. mRNA from cultured fibroblasts obtained from a normal control and from the propositus was reverse transcribed. The cDNAs coding for Hex A {alpha}-chain were amplified in four overlapping fragments. In the patient sample it was not possible to amplify the fragment containing the exon 11/intron 11 junction, indicating that this mutation alters normal RNA processing of the Hex A pre-mRNA resulting in the deficiency of Hex A activity.

  15. A Microfabricated Platform for Generating Physiologically-Relevant Hepatocyte Zonation

    PubMed Central

    McCarty, William J.; Usta, O. Berk; Yarmush, Martin L.

    2016-01-01

    In vitro liver models have been important tools for more than 40 years for academic research and preclinical toxicity screening by the pharmaceutical industry. Hepatocytes, the highly metabolic parenchymal cells of the liver, are efficient at different metabolic chemistries depending on their relative spatial location along the sinusoid from the portal triad to the central vein. Although replicating hepatocyte metabolic zonation is vitally important for physiologically-relevant in vitro liver tissue and organ models, it is most often completely overlooked. Here, we demonstrate the creation of spatially-controlled zonation across multiple hepatocyte metabolism levels through the application of precise concentration gradients of exogenous hormone (insulin and glucagon) and chemical (3-methylcholanthrene) induction agents in a microfluidic device. Observed gradients in glycogen storage via periodic acid-Schiff staining, urea production via carbamoyl phosphatase synthetase I staining, and cell viability after exposure to allyl alcohol and acetaminophen demonstrated the in vitro creation of hepatocyte carbohydrate, nitrogen, alcohol degradation, and drug conjugation metabolic zonation. This type of advanced control system will be crucial for studies evaluating drug metabolism and toxicology using in vitro constructs. PMID:27240736

  16. Preservation of hepatocyte suspensions at 4 degrees C.

    PubMed

    Lund, P; Wiggins, D

    1988-10-01

    Suspensions of rat hepatocytes are resistant to hypoxia for at least 24 hr at 4 degrees C. After storage for 24 hr with xylitol, sorbitol, or glycerol, their subsequent capacity for glucogenesis from these substrates is increased. Even storage under N2/CO2 as the gas phase has little deleterious effect.

  17. A Microfabricated Platform for Generating Physiologically-Relevant Hepatocyte Zonation

    NASA Astrophysics Data System (ADS)

    McCarty, William J.; Usta, O. Berk; Yarmush, Martin L.

    2016-05-01

    In vitro liver models have been important tools for more than 40 years for academic research and preclinical toxicity screening by the pharmaceutical industry. Hepatocytes, the highly metabolic parenchymal cells of the liver, are efficient at different metabolic chemistries depending on their relative spatial location along the sinusoid from the portal triad to the central vein. Although replicating hepatocyte metabolic zonation is vitally important for physiologically-relevant in vitro liver tissue and organ models, it is most often completely overlooked. Here, we demonstrate the creation of spatially-controlled zonation across multiple hepatocyte metabolism levels through the application of precise concentration gradients of exogenous hormone (insulin and glucagon) and chemical (3-methylcholanthrene) induction agents in a microfluidic device. Observed gradients in glycogen storage via periodic acid-Schiff staining, urea production via carbamoyl phosphatase synthetase I staining, and cell viability after exposure to allyl alcohol and acetaminophen demonstrated the in vitro creation of hepatocyte carbohydrate, nitrogen, alcohol degradation, and drug conjugation metabolic zonation. This type of advanced control system will be crucial for studies evaluating drug metabolism and toxicology using in vitro constructs.

  18. Aniline Induces Oxidative Stress and Apoptosis of Primary Cultured Hepatocytes.

    PubMed

    Wang, Yue; Gao, Hong; Na, Xiao-Lin; Dong, Shu-Ying; Dong, Hong-Wei; Yu, Jia; Jia, Li; Wu, Yong-Hui

    2016-11-30

    The toxicity and carcinogenicity of aniline in humans and animals have been well documented. However, the molecular mechanism involved in aniline-induced liver toxicity and carcinogenesis remains unclear. In our research, primary cultured hepatocytes were exposed to aniline (0, 1.25, 2.50, 5.0 and 10.0 μg/mL) for 24 h in the presence or absence of N-acetyl-l-cysteine (NAC). Levels of reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH), activities of superoxide dismutase (SOD) and catalase (CAT), mitochondrial membrane potential, DNA damage, cell viability, and apoptosis were detected. Levels of ROS and MDA were significantly increased and levels of GSH and CAT, activity of SOD, and mitochondrial membrane potential in hepatocytes were significantly decreased by aniline compared with the negative control group. The tail moment and DNA content of the tail in exposed groups were significantly higher than those in the negative control group. Cell viability was reduced and apoptotic death was induced by aniline in a concentration-dependent manner. The phenomena of ROS generation, oxidative damage, loss of mitochondrial membrane potential, DNA damage and apoptosis could be prevented if ROS inhibitor NAC was added. ROS generation is involved in the loss of mitochondrial membrane potential and DNA injury, which may play a role in aniline-induced apoptosis in hepatocytes. Our study provides insight into the mechanism of aniline-induced toxicity and apoptosis of hepatocytes.

  19. Liver glycogen bodies: ground-glass hepatocytes in transplanted patients.

    PubMed

    Bejarano, Pablo A; Garcia, Monica T; Rodriguez, Maria M; Ruiz, Phillip; Tzakis, Andreas G

    2006-11-01

    Ground-glass hepatocytes have been described in Lafora's disease, fibrinogen deposition, hepatitis B, type IV glycogenosis, and alcohol aversion (cyanamide) therapy. We encountered ground-glass hepatocytes with intracytoplasmic inclusions in four liver biopsies from three transplanted patients who had none of the above-mentioned underlying diseases. One patient was a 4-year-old boy who had a kidney transplant for severe ureterovesical reflux. Patient 2 was a 52-year-old man who had two liver transplants because of hepatitis C. The third patient was a 7-month-old girl who underwent a multivisceral transplant because of necrotizing enterocolitis and liver failure induced by total parenteral nutrition. The patients developed liver abnormalities from 45 days to 4 years after their transplants. The livers showed conspicuous ground-glass hepatocytes in 90% of the children's samples and 30% of the adult liver cells. The cytoplasmic bodies stained strongly for Gomori methenamine-silver; they were positive for periodic acid-Schiff without diastase, but negative after diastase digestion. They were negative for colloidal iron and hepatitis B core and surface antigens. Electron microscopy revealed non-membrane bound aggregates of glycogen. Idiopathic ground-glass hepatocytes occur in transplanted patients and represent accumulation of altered glycogen. However, their clinical significance and cause are not entirely elucidated.

  20. Octylphenol induces vitellogenin production and cell death in fish hepatocytes

    SciTech Connect

    Toomey, B.H.; Monteverdi, G.H.; Di Giulio, R.T.

    1999-04-01

    The effects of octylphenol (OP) on vitellogenin production and cell death in hepatocytes from brown bullhead catfish (Americurus nebulosus) were studied. Production of vitellogenin was induced in hepatocytes exposed to 10 to 50 {micro}M OP, whereas a higher concentration of OP (100 {micro}M) induced apoptotic cell death. By 3 h after the addition of 100 {micro}M OP, dying cells showed chromatin condensation and DNA fragmentation as determined by fluorescence microscopy and gel electrophoresis. Later stages of cell death (nuclear membrane breakdown and cell fragmentation into apoptotic bodies) were identified in cells exposed to OP for at least 6 h. Hepatocytes exposed to 100 {micro}M OP also produced less vitellogenin than cells exposed to 50 {micro}M OP. An estrogen receptor antagonist, tamoxifen, greatly decreased vitellogenin production in OP-exposed hepatocytes from male fish but did not decrease cell death in these cells. Thus, although the ability of OP to induce vitellogenin production is likely mediated through interactions with the estrogen receptor, the induction of apoptotic cell death by OP does not appear to be dependent on its estrogenic activity but may be a more general toxic effect.

  1. INTERINDIVIDUAL VARIATION IN THE METABOLISM OF ARSENIC IN HUMAN HEPATOCYTES

    EPA Science Inventory


    The liver is the major site for the enzymatic methylation of inorganic arsenic (iAs) in humans. Primary cultures of normal human hepatocytes isolated from tissue obtained at surgery or from donor livers have been used to study interindividual variation in the capacity of live...

  2. 3D Cultivation Techniques for Primary Human Hepatocytes

    PubMed Central

    Bachmann, Anastasia; Moll, Matthias; Gottwald, Eric; Nies, Cordula; Zantl, Roman; Wagner, Helga; Burkhardt, Britta; Sánchez, Juan J. Martínez; Ladurner, Ruth; Thasler, Wolfgang; Damm, Georg; Nussler, Andreas K.

    2015-01-01

    One of the main challenges in drug development is the prediction of in vivo toxicity based on in vitro data. The standard cultivation system for primary human hepatocytes is based on monolayer cultures, even if it is known that these conditions result in a loss of hepatocyte morphology and of liver-specific functions, such as drug-metabolizing enzymes and transporters. As it has been demonstrated that hepatocytes embedded between two sheets of collagen maintain their function, various hydrogels and scaffolds for the 3D cultivation of hepatocytes have been developed. To further improve or maintain hepatic functions, 3D cultivation has been combined with perfusion. In this manuscript, we discuss the benefits and drawbacks of different 3D microfluidic devices. For most systems that are currently available, the main issues are the requirement of large cell numbers, the low throughput, and expensive equipment, which render these devices unattractive for research and the drug-developing industry. A higher acceptance of these devices could be achieved by their simplification and their compatibility with high-throughput, as both aspects are of major importance for a user-friendly device. PMID:27600213

  3. DIFFERENTIATING MECHANISMS OF REACTIVE CHEMICAL TOXICITY IN ISOLATED TROUT HEPATOCYTES

    EPA Science Inventory

    The toxicity of four quinones, 2,3-dimethoxy-1,4-naphthoquinone (DMONQ), 2-methyl 1,4-naphthoquinone (MNQ ),1,4-naphthoquinone (NQ), and 1,4-benzoquinone (BQ), which redox cycle or arlyate in mammalian cells, was determined in isolated trout (Oncorhynchus mykiss) hepatocytes. Mor...

  4. Retinoic Acid-mediated Nuclear Receptor Activation and Hepatocyte Proliferation

    PubMed Central

    Bushue, Nathan; Wan, Yu-Jui Yvonne

    2016-01-01

    Due to their well-known differentiation and apoptosis-inducing abilities, retinoic acid (RA) and its analogs have strong anti-cancer efficacy in human cancers. However, in vivo RA is a liver mitogen. While speculation has persisted that RA-mediated signaling is likely involved in hepatocyte proliferation during liver regeneration, direct evidence is still required. Findings in support of this proposition include observations that a release of retinyl palmitate (the precursor of RA) occurs in liver stellate cells following liver injury. Nevertheless, the biological action of this released vitamin A is virtually unknown. More likely is that the released vitamin A is converted to RA, the biological form, and then bound to a specific receptor (retinoid x receptor; RXRα), which is most abundantly expressed in the liver. Considering the mitogenic effects of RA, the RA-activated RXRα would likely then influence hepatocyte proliferation and liver tissue repair. At present, the mechanism by which RA stimulates hepatocyte proliferation is largely unknown. This review summarizes the activation of nuclear receptors (peroxisome proliferator activated receptor-α, pregnane x receptor, constitutive androstane receptor, and farnesoid x receptor) in an RXRα dependent manner to induce hepatocyte proliferation, providing a link between RA and its proliferative role. PMID:27635169

  5. Asialoglycoprotein receptor mediated hepatocyte targeting - strategies and applications.

    PubMed

    D'Souza, Anisha A; Devarajan, Padma V

    2015-04-10

    Hepatocyte resident afflictions continue to affect the human population unabated. The asialoglycoprotein receptor (ASGPR) is primarily expressed on hepatocytes and minimally on extra-hepatic cells. This makes it specifically attractive for receptor-mediated drug delivery with minimum concerns of toxicity. ASGPR facilitates internalization by clathrin-mediated endocytosis and exhibits high affinity for carbohydrates specifically galactose, N-acetylgalactosamine and glucose. Isomeric forms of sugar, galactose density and branching, spatial geometry and galactose linkages are key factors influencing ligand-receptor binding. Popular ligands for ASGPR mediated targeting are carbohydrate polymers, arabinogalactan and pullulan. Other ligands include galactose-bearing glycoproteins, glycopeptides and galactose modified polymers and lipids. Drug-ligand conjugates provide a viable strategy; nevertheless ligand-anchored nanocarriers provide an attractive option for ASGPR targeted delivery and are widely explored. The present review details various ligands and nanocarriers exploited for ASGPR mediated delivery of drugs to hepatocytes. Nanocarrier properties affecting ASGPR mediated uptake are discussed at length. The review also highlights the clinical relevance of ASGPR mediated targeting and applications in diagnostics. ASGPR mediated hepatocyte targeting provides great promise for improved therapy of hepatic afflictions.

  6. Aniline Induces Oxidative Stress and Apoptosis of Primary Cultured Hepatocytes

    PubMed Central

    Wang, Yue; Gao, Hong; Na, Xiao-Lin; Dong, Shu-Ying; Dong, Hong-Wei; Yu, Jia; Jia, Li; Wu, Yong-Hui

    2016-01-01

    The toxicity and carcinogenicity of aniline in humans and animals have been well documented. However, the molecular mechanism involved in aniline-induced liver toxicity and carcinogenesis remains unclear. In our research, primary cultured hepatocytes were exposed to aniline (0, 1.25, 2.50, 5.0 and 10.0 μg/mL) for 24 h in the presence or absence of N-acetyl-l-cysteine (NAC). Levels of reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH), activities of superoxide dismutase (SOD) and catalase (CAT), mitochondrial membrane potential, DNA damage, cell viability, and apoptosis were detected. Levels of ROS and MDA were significantly increased and levels of GSH and CAT, activity of SOD, and mitochondrial membrane potential in hepatocytes were significantly decreased by aniline compared with the negative control group. The tail moment and DNA content of the tail in exposed groups were significantly higher than those in the negative control group. Cell viability was reduced and apoptotic death was induced by aniline in a concentration-dependent manner. The phenomena of ROS generation, oxidative damage, loss of mitochondrial membrane potential, DNA damage and apoptosis could be prevented if ROS inhibitor NAC was added. ROS generation is involved in the loss of mitochondrial membrane potential and DNA injury, which may play a role in aniline-induced apoptosis in hepatocytes. Our study provides insight into the mechanism of aniline-induced toxicity and apoptosis of hepatocytes. PMID:27916916

  7. TEMPORAL CHANGE IN GAP JUNCTION FUNCTION IN PRIMARY HEPATOCYTES

    EPA Science Inventory

    TEMPORAL CHANGES IN GAP JUNCTION FUNCTION IN PRIMARY *

    The objective of this study was to examine the reduction in gap junction communication (GJC) in primary hepatocytes due to coincident melatonin and magnetic field treatments to determine if these conditions could prov...

  8. Lipid droplet breakdown requires Dynamin 2 for vesiculation of autolysosomal tubules in hepatocytes

    PubMed Central

    Schulze, Ryan J.; Weller, Shaun G.; Schroeder, Barbara; Krueger, Eugene W.; Chi, Susan; Casey, Carol A.

    2013-01-01

    Lipid droplets (LDs) are lipid storage organelles that in hepatocytes may be catabolized by autophagy for use as an energy source, but the membrane-trafficking machinery regulating such a process is poorly characterized. We hypothesized that the large GTPase Dynamin 2 (Dyn2), well known for its involvement in membrane deformation and cellular protein trafficking, could orchestrate autophagy-mediated LD breakdown. Accordingly, depletion or pharmacologic inhibition of Dyn2 led to a substantial accumulation of LDs in hepatocytes. Strikingly, the targeted disruption of Dyn2 induced a dramatic four- to fivefold increase in the size of autolysosomes. Chronic or acute Dyn2 inhibition combined with nutrient deprivation stimulated the excessive tubulation of these autolysosomal compartments. Importantly, Dyn2 associated with these tubules along their length, and the tubules vesiculated and fragmented in the presence of functional Dyn2. These findings provide new evidence for the participation of the autolysosome in LD metabolism and demonstrate a novel role for dynamin in the function and maturation of an autophagic compartment. PMID:24145164

  9. Propionate induces mRNA expression of gluconeogenic genes in bovine calf hepatocytes.

    PubMed

    Zhang, Qian; Koser, Stephanie L; Donkin, Shawn S

    2016-05-01

    Hepatocytes monolayers from neonatal calves were used to determine the responses of the cytosolic phosphoenolpyruvate carboxykinase (PCK1) mRNA expression to propionate and direct hormonal cues including cyclic AMP (cAMP), dexamethasone, and insulin. The responses of other key gluconeogenic genes, including mitochondrial phosphoenolpyruvate carboxykinase (PCK2), pyruvate carboxylase (PC), and glucose-6-phosphotase (G6PC), were also measured. Expression of PCK1 was linearly induced with increasing propionate concentrations in media and 2.5 mM propionate increased PCK1 mRNA at 3 and 6h of incubation; however, the induction disappeared at 12 and 24 h. The induction of PCK1 mRNA by propionate was mimicked by 1 mM cAMP, or in combination with 5 µM dexamethasone, but not by dexamethasone alone. The induction of PCK1 mRNA by propionate or cAMP was eliminated by addition of 100 nM insulin. Additionally, expression of PCK2 and PC mRNA was also induced by propionate in a concentration-dependent manner. Consistent with PCK1, propionate-stimulated PCK2 and PC mRNA expression was inhibited by insulin. Expression of G6PC mRNA was neither affected by propionate nor cAMP, dexamethasone, insulin, or their combinations. These findings demonstrate that propionate can directly regulate its own metabolism in bovine calf hepatocytes through upregulation of PCK1, PCK2, and PC mRNA expression.

  10. Possible mechanism for hepatitis B virus X gene to induce apoptosis of hepatocytes

    PubMed Central

    Zhang, Sheng-Jun; Chen, Hong-Ying; Chen, Zhi-Xin; Wang, Xiao-Zhong

    2005-01-01

    AIM: To investigate the possible mechanism for HBV X gene to induce apoptosis of hepatocyte HL-7702 cells. METHODS: HBV X gene eukaryon expression vector pcDNA3-X was established and transfected into HL-7702 cells by lipid-mediated transfection, including transient and stable transfection. Positive clones were screened by incubating in the selective medium with 600 mg/mL G418 and named HL-7702/HBV-encoded X protein (HBx) cells. The expressions of Fas/FasL, Bax/Bcl-2, and c-myc mRNA were measured by semi-quantitative RT-PCR in HL-7702/HBx and control group, respectively. RESULTS: RT-PCR analysis confirmed that HBV X gene was transfected into HL-7702 cells successfully. By semi-quantitative RT-PCR analysis, Bax and c-myc mRNA levels in HL-7702/HBx cells of transient transfection were significantly higher than those in control, FasL and c-myc mRNA levels in HL-7702/HBx cells of stable transfection were significantly higher than those in control, whereas the Bcl-2 mRNA levels in HL-7702/HBx cells of transient and stable transfection were significantly lower than those in control. CONCLUSION: HBV X gene may promote the apoptosis of hepatocytes by regulating the expressions of Fas/FasL, Bax/Bcl-2, and c-myc gene in a dose-dependent manner. PMID:16038033

  11. Natural pyrethrins induces apoptosis in human hepatocyte cells via Bax- and Bcl-2-mediated mitochondrial pathway.

    PubMed

    Yang, Yun; Zong, Mimi; Xu, Wenping; Zhang, Yang; Wang, Bo; Yang, Mingjun; Tao, Liming

    2017-01-25

    Natural pyrethrins have been widely used for pest control in organic farming and for residential indoor pest managements. Although the specific mechanisms underlying their activity are incompletely understood, natural pesticides are considered the safest based on their target specificity and rapid degradation in the environment. Here, we used in vitro bioassays to characterize the cytotoxic effects of natural pyrethrins and attempted to delineate the cellular and molecular mechanisms of their cytotoxicity against human hepatocytes. The results demonstrate that natural pyrethrins reduce cell viability and enhance apoptosis in HepG2 cells. In addition, the current data indicate that natural pyrethrins cause a reduction in the mitochondrial membrane potential (Δψm), increase reactive oxygen species production, and up-regulate the Bax/Bcl-2 expression, leading to the release of cytochrome-c into the cytosol, activation of caspase-9 and caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP). Taken together, the results indicate that natural pyrethrins has potentially exert adverse effects on human health by inducing caspase-dependent apoptosis in hepatocytes through Bax- and Bcl-2-mediated mitochondrial pathway.

  12. Clustering Nuclear Receptors in Liver Regeneration Identifies Candidate Modulators of Hepatocyte Proliferation and Hepatocarcinoma

    PubMed Central

    Graziano, Giusi; D'Orazio, Andria; Cariello, Marica; Massafra, Vittoria; Salvatore, Lorena; Martelli, Nicola; Murzilli, Stefania; Sasso, Giuseppe Lo; Mariani-Costantini, Renato; Moschetta, Antonio

    2014-01-01

    Background & Aims Liver regeneration (LR) is a valuable model for studying mechanisms modulating hepatocyte proliferation. Nuclear receptors (NRs) are key players in the control of cellular functions, being ideal modulators of hepatic proliferation and carcinogenesis. Methods & Results We used a previously validated RT-qPCR platform to profile modifications in the expression of all 49 members of the NR superfamily in mouse liver during LR. Twenty-nine NR transcripts were significantly modified in their expression during LR, including fatty acid (peroxisome proliferator-activated receptors, PPARs) and oxysterol (liver X receptors, Lxrs) sensors, circadian masters RevErbα and RevErbβ, glucocorticoid receptor (Gr) and constitutive androxane receptor (Car). In order to detect the NRs that better characterize proliferative status vs. proliferating liver, we used the novel Random Forest (RF) analysis to selected a trio of down-regulated NRs (thyroid receptor alpha, Trα; farsenoid X receptor beta, Fxrβ; Pparδ) as best discriminators of the proliferating status. To validate our approach, we further studied PPARδ role in modulating hepatic proliferation. We first confirmed the suppression of PPARδ both in LR and human hepatocellular carcinoma at protein level, and then demonstrated that PPARδ agonist GW501516 reduces the proliferative potential of hepatoma cells. Conclusions Our data suggest that NR transcriptome is modulated in proliferating liver and is a source of biomarkers and bona fide pharmacological targets for the management of liver disease affecting hepatocyte proliferation. PMID:25116592

  13. [Use of xenogenic lyophilized hepatocytes in the treatment of acute and chronic liver diseases].

    PubMed

    Musselius, S G; Vasina, N V; Gladskikh, L V

    1998-01-01

    Therapeutic effect of lyophilized xenogenic hepatocytes was demonstrated on 30 dogs with acute hepatic failure and on white mice. The major biochemical values were corrected and the yeast fermentation test showed biological activity of isolated hepatocytes. Clinically, lyophilized hepatocytes were used in the treatment of patients with acute hepatic failure: orally in 47 and for extracorporeal dialysis in 8. The therapeutic effect of lyophilized hepatocytes is based on active detoxication and hemostasis correction. Clinical, laboratory, and instrumental studies showed improvement of the clinical status, decreased encephalopathy, and accelerated repair processes in the liver. Addition of lyophilized hepatocytes to combined therapy decreased the mortality by 2.5 times.

  14. Enzymatic antioxidant defense in isolated rat hepatocytes exposed to cadmium.

    PubMed

    Skrzycki, M; Czeczot, H; Majewska, M; Podsiad, M; Karlik, W; Grono, D; Wiechetek, M

    2010-01-01

    The aim of the study was the evaluation of cadmium effects on the activity of antioxidant enzymes in rat hepatocytes. The studies were conducted with isolated rat hepatocytes incubated for 1 or 2 hours in a modified (deprived of carbonates with phosphates) Williams' E medium (MWE) in the presence of cadmium chloride (25, 50 and 200 microM). Hepatocytes incubated in the MWE medium without cadmium chloride were used as a control. The application of the modified Williams' E medium allowed for the appearance of cadmium compounds in a soluble form that is indispensable for suitable estimation of its toxic action. There were evaluated markers of the oxidative stress such as: concentration of thiobarbiturate reactive substances (TBARS)--proportional to the level of lipid peroxidation, concentration of reduced glutathione (GSH), and the activity of antioxidant enzymes, including superoxide dismutase (SOD1 and SOD2), catalase (CAT), total glutathione peroxidase (GSHPx), selenium--dependent glutathione peroxidase (SeGSHPx), glutathione transferase (GST) and glutathione reductase (GSHR). Alterations of antioxidant enzymes activity, the level of TBARS and GSH in isolated rat hepatocytes caused by cadmium in vitro, were shown to depend on the concentration and time of exposure of cells to this metal. The increased level of TBARS and GSH was observed as well as changes in the activity of antioxidant enzymes. The activity of SOD isoenzymes and CAT was increased, whereas GSHPx and GST were decreased. These results indicate that cadmium induces oxidative stress followed by alterations in the cellular antioxidant enzyme system in isolated rat hepatocytes.

  15. Epigallocatechin-3-gallate ameliorates insulin resistance in hepatocytes.

    PubMed

    Ma, Shan-Bo; Zhang, Rui; Miao, Shan; Gao, Bin; Lu, Yang; Hui, Sen; Li, Long; Shi, Xiao-Peng; Wen, Ai-Dong

    2017-04-07

    Hyperglycemia is a typical pathogenic factor in a series of complications among patients with type II diabetes. Epigallocatechin-3-gallate (EGCG) is the major polyphenol extracted from green tea and is reported to be an antioxidant. The aim of the present study was to examine the effect of EGCG on insulin resistance in human HepG2 cells pretreated with high concentrations of glucose. The protein kinase B (AKT)/glycogen synthase kinase (GSK) pathways were analyzed using western blot analysis in HepG2 cells and primary mouse hepatocytes treated with high glucose and/or EGCG. Cellular glycogen content was determined using a glycogen assay kit. Reactive oxygen species (ROS) production was determined using dihydroethidium staining and flow cytometry. c‑JUN N‑terminal kinase (JNK)/insulin receptor substrate 1 (IRS1)/AKT/GSK signaling was explored using western blot analysis in HepG2 cells treated with high glucose and/or EGCG or N-acetyl-cysteine. High glucose significantly decreased the levels of phosphorylated AKT and GSK in HepG2 cells and mouse primary hepatocytes. Pretreatment with EGCG significantly restored the activation of AKT and GSK in HepG2 cells and primary hepatocytes exposed to high glucose. In HepG2 cells and primary hepatocytes, glycogen synthesis was improved by EGCG treatment in a dose‑dependent manner. High glucose significantly stimulated the production of ROS while EGCG protected high glucose‑induced ROS production. ROS is known to serve a major role in high glucose induced‑insulin resistance by increasing JNK and IRS1 serine phosphorylation. In the present study, EGCG was observed to enhance the insulin‑signaling pathway. EGCG ameliorated high glucose‑induced insulin resistance in the hepatocytes by potentially decreasing ROS‑induced JNK/IRS1/AKT/GSK signaling.

  16. Epidermal growth factor-stimulated protein phosphorylation in rat hepatocytes

    SciTech Connect

    Connelly, P.A.; Sisk, R.B.; Johnson, R.M.; Garrison, J.C.

    1987-05-01

    Epidermal growth factor (EGF) causes a 6-fold increase in the phosphorylation state of a cytosolic protein (pp36, M/sub r/ = 36,000, pI = 5.5) in hepatocytes isolated from fasted, male, Wistar rats. Stimulation of /sup 32/P incorporation is observed as early as 1 min following treatment of hepatocytes with EGF and is still present at 30 min after exposure to the growth factor. The phosphate incorporated into pp36 in response to EGF is located predominantly in serine but not tyrosine residues. Phosphorylation of pp36 does not occur in response to insulin or to agents which specifically activate the cAMP-dependent protein kinase (S/sub p/ -cAMPS), protein kinase C (PMA) or Ca/sup 2 +//calmodulin-dependent protein kinases (A23187) in these cells. Prior treatment of hepatocytes with the cAMP analog, S/sub p/-cAMPS, or ADP-ribosylation of N/sub i/, the inhibitory GTP-binding protein of the adenylate cyclase complex, does not prevent EGF-stimulated phosphorylation of pp36. However, as seen in other cell types, pretreatment of hepatocytes with PMA abolishes all EGF-mediated responses including phosphorylation of pp36. These results suggest that EGP specifically activates an uncharacterized, serine protein kinase in hepatocytes that is distal to the intrinsic EGF receptor tyrosine protein kinase. The rapid activation of this kinase suggests that it may play an important role in the early response of the cell to EGF.

  17. Augmenter of liver regeneration (ALR) protects human hepatocytes against apoptosis

    SciTech Connect

    Ilowski, Maren; Kleespies, Axel; Toni, Enrico N. de; Donabauer, Barbara; Jauch, Karl-Walter; Hengstler, Jan G.; Thasler, Wolfgang E.

    2011-01-07

    Research highlights: {yields} ALR decreases cytochrome c release from mitochondria. {yields} ALR protects hepatocytes against apoptosis induction by ethanol, TRAIL, anti-Apo, TGF-{beta} and actinomycin D. {yields} ALR exerts a liver-specific anti-apoptotic effect. {yields} A possible medical usage of ALR regarding protection of liver cells during apoptosis inducing therapies. -- Abstract: Augmenter of liver regeneration (ALR) is known to support liver regeneration and to stimulate proliferation of hepatocytes. However, it is not known if ALR exerts anti-apoptotic effects in human hepatocytes and whether this protective effect is cell type specific. This is relevant, because compounds that protect the liver against apoptosis without undesired effects, such as protection of metastatic tumour cells, would be appreciated in several clinical settings. Primary human hepatocytes (phH) and organotypic cancer cell lines were exposed to different concentrations of apoptosis inducers (ethanol, TRAIL, anti-Apo, TGF-{beta}, actinomycin D) and cultured with or without recombinant human ALR (rhALR). Apoptosis was evaluated by the release of cytochrome c from mitochondria and by FACS with propidium iodide (PI) staining. ALR significantly decreased apoptosis induced by ethanol, TRAIL, anti-Apo, TGF-{beta} and actinomycin D. Further, the anti-apoptotic effect of ALR was observed in primary human hepatocytes and in HepG2 cells but not in bronchial (BC1), colonic (SW480), gastric (GC1) and pancreatic (L3.6PL) cell lines. Therefore, the hepatotrophic growth factor ALR acts in a liver specific manner with regards to both its mitogenic and its anti-apoptotic effect. Unlike the growth factors HGF and EGF, rhALR acts in a liver specific manner. Therefore, ALR is a promising candidate for further evaluation as a possible hepatoprotective factor in clinical settings.

  18. Characterization of aldehyde oxidase enzyme activity in cryopreserved human hepatocytes.

    PubMed

    Hutzler, J Matthew; Yang, Young-Sun; Albaugh, Daniel; Fullenwider, Cody L; Schmenk, Jennifer; Fisher, Michael B

    2012-02-01

    Substrates of aldehyde oxidase (AO), for which human clinical pharmacokinetics are reported, were selected and evaluated in pooled mixed-gender cryopreserved human hepatocytes in an effort to quantitatively characterize AO activity. Estimated hepatic clearance (Cl(h)) for BIBX1382, carbazeran, O⁶-benzylguanine, zaleplon, and XK-469 using cryopreserved hepatocytes was 18, 17, 12, <4.3, and <4.3 ml · min⁻¹ · kg⁻¹, respectively. The observed metabolic clearance in cryopreserved hepatocytes was confirmed to be a result of AO-mediated metabolism via two approaches. Metabolite identification after incubations in the presence of H₂¹⁸O confirmed that the predominant oxidative metabolite was generated by AO, as expected isotope patterns in mass spectra were observed after analysis by high-resolution mass spectrometry. Second, clearance values were efficiently attenuated upon coincubation with hydralazine, an inhibitor of AO. The low exposure after oral doses of BIBX1382 and carbazeran (∼5% F) would have been fairly well predicted using simple hepatic extraction (f(h)) values derived from cryopreserved hepatocytes. In addition, the estimated hepatic clearance value for O⁶-benzylguanine was within ∼80% of the observed total clearance in humans after intravenous administration (15 ml · min⁻¹ · kg⁻¹), indicating a reasonable level of quantitative activity from this in vitro system. However, a 3.5-fold underprediction of total clearance was observed for zaleplon, despite the 5-oxo metabolite being clearly observed. These data taken together suggest that the use of cryopreserved hepatocytes may be a practical approach for assessing AO-mediated metabolism in discovery and potentially useful for predicting hepatic clearance of AO substrates.

  19. Microcystin-LR induces anoikis resistance to the hepatocyte uptake transporter OATP1B3-expressing cell lines.

    PubMed

    Takano, Hiroyuki; Takumi, Shota; Ikema, Satoshi; Mizoue, Nozomi; Hotta, Yuki; Shiozaki, Kazuhiro; Sugiyama, Yasumasa; Furukawa, Tatsuhiko; Komatsu, Masaharu

    2014-12-04

    Microcystin-LR is a cyclic peptide released by several bloom-forming cyanobacteria. Understanding the mechanism of microcystin-LR toxicity is important, because of the both potencies of its acute cytotoxicity and tumor-promoting activity in hepatocytes of animals and humans. Recently, we have reported that the expression of human hepatocyte uptake transporter OATP1B3 was critical for the selective uptake of microcystin-LR into hepatocytes and for induction of its fatal cytotoxicity. In this study, we demonstrated a novel function of microcystin-LR which induced bipotential changes including anoikis resistance and cytoskeleton reorganization to OATP1B3-transfected HEK293 cells (HEK293-OATP1B3). After exposure to microcystin-LR, HEK293-OATP1B3 cells were divided to the floating cells and remaining adherent cells. After collection and reseeding the floating cells into a fresh flask, cells were confluently proliferated (HEK293-OATP1B3-FL) under the microcystin-LR-free condition. Both the proliferated HEK293-OATP1B3-FL and remaining adherent HEK293-OATP1B3-AD cells changed the character with down- and up-regulation of E-cadherin, respectively. Additionally, these cells acquired resistance to microcystin-LR. These results suggest that microcystin-LR could be associated with not only tumor promotion, but also epithelial-mesenchymal transition-mediated cancer metastasis. Furthermore, microcystin-LR might induce the cytoskeleton reorganization be accompanied epithelial-mesenchymal transition.

  20. Hepatocyte-secreted extracellular vesicles modify blood metabolome and endothelial function by an arginase-dependent mechanism

    PubMed Central

    Royo, Felix; Moreno, Laura; Mleczko, Justyna; Palomo, Laura; Gonzalez, Esperanza; Cabrera, Diana; Cogolludo, Angel; Vizcaino, Francisco Perez; van-Liempd, Sebastiaan; Falcon-Perez, Juan M.

    2017-01-01

    Hepatocytes release extracellular vesicles (EVs) loaded with signaling molecules and enzymes into the bloodstream. Although the importance of EVs in the intercellular communication is already recognized, the metabolic impact of the enzymes carried by these vesicles is still unclear. We evaluated the global effect of the enzymatic activities of EVs by performing untargeted metabolomic profiling of serum samples after their exposure to EVs. This approach revealed a significant change in the abundance of 94 serum metabolic signals. Our study shows that these vesicles modify the concentration of metabolites of different chemical nature including metabolites related to arginine metabolism, which regulates vascular function. To assess the functional relevance of this finding, we examined the levels of arginase-1 protein and its activity in the hepatic EVs carrying the exosomal markers CD81 and CD63. Remarkably, the arginase activity was also detected in EVs isolated from the serum in vivo, and this vesicular activity significantly increased under liver-damaging conditions. Finally, we demonstrated that EVs secreted by hepatocytes inhibited the acetylcholine-induced relaxation in isolated pulmonary arteries, via an arginase-dependent mechanism. In summary, our study demonstrates that the hepatocyte-released EVs are metabolically active, affecting a number of serum metabolites involved in oxidative stress metabolism and the endothelial function. PMID:28211494

  1. Hepatocytes release ceramide-enriched pro-inflammatory extracellular vesicles in an IRE1α-dependent manner[S

    PubMed Central

    Kakazu, Eiji; Mauer, Amy S.; Yin, Meng; Malhi, Harmeet

    2016-01-01

    Nonalcoholic steatohepatitis (NASH) is a lipotoxic disease wherein activation of endoplasmic reticulum (ER) stress response and macrophage-mediated hepatic inflammation are key pathogenic features. However, the lipid mediators linking these two observations remain elusive. We postulated that ER stress-regulated release of pro-inflammatory extracellular vesicles (EVs) from lipotoxic hepatocytes may be this link. EVs were isolated from cell culture supernatants of hepatocytes treated with palmitate (PA) to induce lipotoxic ER stress, characterized by immunofluorescence, Western blotting, electron microscopy, and nanoparticle tracking analysis. Sphingolipids were measured by tandem mass spectrometry. EVs were employed in macrophage chemotaxis assays. PA induced significant EV release. Because PA activates ER stress, we used KO hepatocytes to demonstrate that PA-induced EV release was mediated by inositol requiring enzyme 1α (IRE1α)/X-box binding protein-1. PA-induced EVs were enriched in C16:0 ceramide in an IRE1α-dependent manner, and activated macrophage chemotaxis via formation of sphingosine-1-phosphate (S1P) from C16:0 ceramide. This chemotaxis was blocked by sphingosine kinase inhibitors and S1P receptor inhibitors. Lastly, elevated circulating EVs in experimental and human NASH demonstrated increased C16:0 ceramide. PA induces C16:0 ceramide-enriched EV release in an IRE1α-dependent manner. The ceramide metabolite, S1P, activates macrophage chemotaxis, a potential mechanism for the recruitment of macrophages to the liver under lipotoxic conditions. PMID:26621917

  2. Amylin impairment of insulin effects on glycogen synthesis and phosphoenolpyruvate carboxykinase gene expression in rat primary cultured hepatocytes.

    PubMed Central

    Baqué, S; Guinovart, J J; Gómez-Foix, A M

    1994-01-01

    The ability of amylin to impair hepatic insulin action is controversial. We have found that the effect of amylin in primary cultured hepatocytes is strongly dependent on the culture conditions. Only in hepatocytes preincubated in the presence of fetal serum did amylin, at concentrations ranging from 1 to 100 nM, reduce insulin-stimulated glycogen synthesis rate and glycogen accumulation without showing direct effects. Neither basal glycogen synthase nor glycogen phosphorylase activity was modified by amylin treatment. Nevertheless, amylin (100 nM) blocked the activation of glycogen synthase by insulin. Amylin also proved capable of opposing the reduction in the expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene induced by insulin, whereas the basal mRNA level of PEPCK was unaffected by amylin treatment. Thus, these results show that, in cultured rat hepatocytes, amylin is indeed able to interfere with insulin regulation of glycogenesis and PEPCK gene expression, favouring the hypothesis that amylin may modulate liver sensitivity to insulin. Images Figure 3 PMID:7998979

  3. Positions of pluripotency genes and hepatocyte-specific genes in the nucleus before and after mouse ES cell differentiation.

    PubMed

    Udagawa, K; Ohyama, T

    2014-03-24

    Spatial positioning of genes in the cell nucleus plays an important role in the regulation of genomic functions. Evidence for changes in gene positioning associated with transcriptional activity has been reported. However, our understanding of this phenomenon is still quite limited. We examined how pluripotency genes and hepatocyte-specific genes behave during the differentiation of mouse embryonic stem (ES) cells into hepatocytes, by targeting the loci of the Klf4, Nanog, Oct4, Sox2, Cyp7α1, Pck1, Tat, and Tdo2 genes, and using three-dimensional fluorescence in situ hybridization analyses. We found that each gene has a distinctly inherent localization profile in the ES cell nucleus. During differentiation, the Klf4, Nanog, Oct4, Cyp7α1, Pck1, and Tat loci shifted toward the nuclear center, while the Sox2 and Tdo2 loci shifted toward the periphery. The Klf4, Nanog, Oct4, and Tdo2 seem to prefer the outer regions, rather than the inner regions, when they are active. We also found that the radial positioning of the focused genes in the hepatocyte cell nucleus was highly correlated with the local GC content and the gene density of the surrounding region, but not with gene activity.

  4. Effects of tert-butyl hydroperoxide on Ca(2+) ATPase activity in isolated rat hepatocytes and its reversal by antioxidants.

    PubMed

    Singh, Sangram; Agarwal, Richa; Jamal, Farrukh; Mehrotra, Sudhir; Singh, Rakesh

    2012-09-01

    Calcium ions play an importantrole in various physiological processes such as nerve impulse transmission, muscle contraction, hormone action, blood clotting. They ions act as an intracellular second messenger, relaying information within cells to regulate their activity. To understand the mechanism of hepatotoxicity of t-BHP, studies were carried out using freshly isolated rat hepatocytes. The effect of t-BHP on Ca(2+) accumulation and Ca(2+) uptake by rat hepatocytes was monitored using 45Ca(2+). It caused decrease in 15% accumulation of 45Ca(2+) in comparison to the control group. t-BHP also significantly decreased the Ca(2+) ATPase activity in isolated hepatocytes .This decrease in Ca(2+) ATPase activity by t-BHP was reversed 40% by naturally occurring antioxidant glutathione (GSH) and 20% by the synthetic antioxidant butylated hydroxy toluene (BHT). These results indicate that the hepatotoxic action of t-BHP involves oxidative stress as evident by the protection accorded by various antioxidants employed in the study as well as impairment of intracellular calcium homeostasis which can lead to liver cell injury.

  5. Comparison of rat and hamster hepatocyte primary culture/DNA repair assays

    SciTech Connect

    Kornbrust, D.J.; Barfknect, T.R.

    1984-01-01

    Previous studies have demonstrated marked differences in the capacity of hepatocytes from rats or hamsters to mediate the metabolic activation of chemical carcinogens to genotoxic (i.e., mutagenic) products. Thus far, very few investigations of species differences in DNA repair have been performed. Therefore, a comparison of the relative extent of DNA repair elicited by various genotoxic chemicals in rat and hamster hepatocyes was conducted, using the hepatocyte primary culture/DNA repair (HPC/DR) assay. Of the ll chemicals tested, eight were more potent in inducing DNA repair in hamster hepatocytes than in rat hepatocytes. Dimethylnitrosamine, diethylnitrosamine, 2-acetylaminofluorene, 9-aminoacridine, pararosaniline hydrochloride, 1-naphthylamine, benzidine and 1,2,3,4-diepoxybutane were all active in hamster hepatocytes at a concentration at least ten times less than the lowest effective concentration in rat hepatocytes. The direct-acting alkylating agent, methylmethane sulfonate, was equipotent inducing DNA repair in both rat and hamster hepatocytes, indicating that the differences in DNA repair observed for the other chemicals were probably not a result of species differences in DNA repair capacities. In contrast, 1-nitropyrene produced a greater DNA repair response in rat hepatocyes than hamster hepatocytes, while the bacterial mutagen 3-(chloromethyl)pyridine hydrochloride was inactive in both hepatocyte systems. These studies demonstrate the feasibility of using hamster hepatocytes in the HPC/DR assay and illustrate the utility of performing the assay with hepatocytes from more than one species.

  6. Directed random walks and constraint programming reveal active pathways in hepatocyte growth factor signaling.

    PubMed

    Kittas, Aristotelis; Delobelle, Aurélien; Schmitt, Sabrina; Breuhahn, Kai; Guziolowski, Carito; Grabe, Niels

    2016-01-01

    An effective means to analyze mRNA expression data is to take advantage of established knowledge from pathway databases, using methods such as pathway-enrichment analyses. However, pathway databases are not case-specific and expression data could be used to infer gene-regulation patterns in the context of specific pathways. In addition, canonical pathways may not always describe the signaling mechanisms properly, because interactions can frequently occur between genes in different pathways. Relatively few methods have been proposed to date for generating and analyzing such networks, preserving the causality between gene interactions and reasoning over the qualitative logic of regulatory effects. We present an algorithm (MCWalk) integrated with a logic programming approach, to discover subgraphs in large-scale signaling networks by random walks in a fully automated pipeline. As an exemplary application, we uncover the signal transduction mechanisms in a gene interaction network describing hepatocyte growth factor-stimulated cell migration and proliferation from gene-expression measured with microarray and RT-qPCR using in-house perturbation experiments in a keratinocyte-fibroblast co-culture. The resulting subgraphs illustrate possible associations of hepatocyte growth factor receptor c-Met nodes, differentially expressed genes and cellular states. Using perturbation experiments and Answer Set programming, we are able to select those which are more consistent with the experimental data. We discover key regulator nodes by measuring the frequency with which they are traversed when connecting signaling between receptors and significantly regulated genes and predict their expression-shift consistently with the measured data. The Java implementation of MCWalk is publicly available under the MIT license at: https://bitbucket.org/akittas/biosubg.

  7. Human Placental Lactogen Induces CYP2E1 Expression via PI 3-Kinase Pathway in Female Human Hepatocytes

    PubMed Central

    Lee, Jin Kyung; Chung, Hye Jin; Fischer, Liam; Fischer, James; Gonzalez, Frank J.

    2014-01-01

    The state of pregnancy is known to alter hepatic drug metabolism. Hormones that rise during pregnancy are potentially responsible for the changes. Here we report the effects of prolactin (PRL), placental lactogen (PL), and growth hormone variant (GH-v) on expression of major hepatic cytochromes P450 expression and a potential molecular mechanism underlying CYP2E1 induction by PL. In female human hepatocytes, PRL and GH-v showed either no effect or small and variable effects on mRNA expression of CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5. On the other hand, PL increased expression level of CYP2E1 mRNA with corresponding increases in CYP2E1 protein and activity levels. Results from hepatocytes and HepaRG cells indicate that PL does not affect the expression or activity of HNF1α, the known transcriptional activator of basal CYP2E1 expression. Furthermore, transient transfection studies and Western blot results showed that STAT signaling, the previously known mediator of PL actions in certain tissues, does not play a role in CYP2E1 induction by PL. A chemical inhibitor of PI3-kinase signaling significantly repressed the CYP2E1 induction by PL in human hepatocytes, suggesting involvement of PI3-kinase pathway in CYP2E1 regulation by PL. CYP2E1-humanized mice did not exhibit enhanced CYP2E1 expression during pregnancy, potentially because of interspecies differences in PL physiology. Taken together, these results indicate that PL induces CYP2E1 expression via PI3-kinase pathway in human hepatocytes. PMID:24408518

  8. Gender differences in methionine accumulation and metabolism in freshly isolated mouse hepatocytes: Potential roles in toxicity

    SciTech Connect

    Dever, Joseph T.; Elfarra, Adnan A.

    2009-05-01

    L-Methionine (Met) is hepatotoxic at high concentrations. Because Met toxicity in freshly isolated mouse hepatocytes is gender-dependent, the goal of this study was to assess the roles of Met accumulation and metabolism in the increased sensitivity of male hepatocytes to Met toxicity compared with female hepatocytes. Male hepatocytes incubated with Met (30 mM) at 37 {sup o}C exhibited higher levels of intracellular Met at 0.5, 1.0, and 1.5 h, respectively, compared to female hepatocytes. Conversely, female hepatocytes had higher levels of S-adenosyl-L-methionine compared to male hepatocytes. Female hepatocytes also exhibited higher L-methionine-L-sulfoxide levels relative to control hepatocytes, whereas the increases in L-methionine-D-sulfoxide (Met-D-O) levels were similar in hepatocytes of both genders. Addition of aminooxyacetic acid (AOAA), an inhibitor of Met transamination, significantly increased Met levels at 1.5 h and increased Met-D-O levels at 1.0 and 1.5 h only in Met-exposed male hepatocytes. No gender differences in cytosolic Met transamination activity by glutamine transaminase K were detected. However, female mouse liver cytosol exhibited higher methionine-DL-sulfoxide (MetO) reductase activity than male mouse liver cytosol at low (0.25 and 0.5 mM) MetO concentrations. Collectively, these results suggest that increased cellular Met accumulation, decreased Met transmethylation, and increased Met and MetO transamination in male mouse hepatocytes may be contributing to the higher sensitivity of the male mouse hepatocytes to Met toxicity in comparison with female mouse hepatocytes.

  9. Gender differences in methionine accumulation and metabolism in freshly isolated mouse hepatocytes: potential roles in toxicity.

    PubMed

    Dever, Joseph T; Elfarra, Adnan A

    2009-05-01

    L-methionine (Met) is hepatotoxic at high concentrations. Because Met toxicity in freshly isolated mouse hepatocytes is gender-dependent, the goal of this study was to assess the roles of Met accumulation and metabolism in the increased sensitivity of male hepatocytes to Met toxicity compared with female hepatocytes. Male hepatocytes incubated with Met (30 mM) at 37 degrees C exhibited higher levels of intracellular Met at 0.5, 1.0, and 1.5 h, respectively, compared to female hepatocytes. Conversely, female hepatocytes had higher levels of S-adenosyl-L-methionine compared to male hepatocytes. Female hepatocytes also exhibited higher L-methionine-L-sulfoxide levels relative to control hepatocytes, whereas the increases in L-methionine-D-sulfoxide (Met-D-O) levels were similar in hepatocytes of both genders. Addition of aminooxyacetic acid (AOAA), an inhibitor of Met transamination, significantly increased Met levels at 1.5 h and increased Met-d-O levels at 1.0 and 1.5 h only in Met-exposed male hepatocytes. No gender differences in cytosolic Met transamination activity by glutamine transaminase K were detected. However, female mouse liver cytosol exhibited higher methionine-dl-sulfoxide (MetO) reductase activity than male mouse liver cytosol at low (0.25 and 0.5 mM) MetO concentrations. Collectively, these results suggest that increased cellular Met accumulation, decreased Met transmethylation, and increased Met and MetO transamination in male mouse hepatocytes may be contributing to the higher sensitivity of the male mouse hepatocytes to Met toxicity in comparison with female mouse hepatocytes.

  10. Rubicon inhibits autophagy and accelerates hepatocyte apoptosis and lipid accumulation in nonalcoholic fatty liver disease in mice.

    PubMed

    Tanaka, Satoshi; Hikita, Hayato; Tatsumi, Tomohide; Sakamori, Ryotaro; Nozaki, Yasutoshi; Sakane, Sadatsugu; Shiode, Yuto; Nakabori, Tasuku; Saito, Yoshinobu; Hiramatsu, Naoki; Tabata, Keisuke; Kawabata, Tsuyoshi; Hamasaki, Maho; Eguchi, Hidetoshi; Nagano, Hiroaki; Yoshimori, Tamotsu; Takehara, Tetsuo

    2016-12-01

    Nonalcoholic fatty liver disease (NAFLD) is the most prevalent liver disease worldwide. It encompasses a spectrum ranging from simple steatosis to fatty liver with hepatocellular injury, termed nonalcoholic steatohepatitis. Recent studies have demonstrated hepatic autophagy being impaired in NAFLD. In the present study, we investigated the impact of Rubicon, a Beclin1-interacting negative regulator for autophagosome-lysosome fusion, in the pathogenesis of NAFLD. In HepG2 cells, BNL-CL2 cells, and murine primary hepatocytes, Rubicon was posttranscriptionally up-regulated by supplementation with saturated fatty acid palmitate. Up-regulation of Rubicon was associated with suppression of the late stage of autophagy, as evidenced by accumulation of both LC3-II and p62 expression levels as well as decreased autophagy flux. Its blockade by small interfering RNA attenuated autophagy impairment and reduced palmitate-induced endoplasmic reticulum stress, apoptosis, and lipid accumulation. Rubicon was also up-regulated in association with autophagy impairment in livers of mice fed a high-fat diet (HFD). Hepatocyte-specific Rubicon knockout mice generated by crossing Rubicon floxed mice with albumin-Cre transgenic mice did not produce any phenotypes on a normal diet. In contrast, on an HFD, they displayed significant improvement of both liver steatosis and injury as well as attenuation of both endoplasmic reticulum stress and autophagy impairment in the liver. In humans, liver tissues obtained from patients with NAFLD expressed significantly higher levels of Rubicon than those without steatosis.

  11. Foxa2 integrates the transcriptional response of the hepatocyte to fasting.

    PubMed

    Zhang, Liping; Rubins, Nir E; Ahima, Rexford S; Greenbaum, Linda E; Kaestner, Klaus H

    2005-08-01

    Survival during prolonged food deprivation depends on the activation of hepatic gluconeogenesis. Inappropriate regulation of this process is a hallmark of diabetes and other metabolic diseases. Activation of the genes encoding gluconeogenic enzymes is mediated by hormone-responsive transcription factors such as the cyclic AMP response element binding protein (CREB) and the glucocorticoid receptor (GR). Here we show using cell-type-specific gene ablation that the winged helix transcription factor Foxa2 is required for activation of the hepatic gluconeogenic program during fasting. Specifically, Foxa2 promotes gene activation both by cyclic AMP, the second messenger for glucagon, and glucocorticoids. Foxa2 mediates these effects by enabling recruitment of CREB and GR to their respective target sites in chromatin. We conclude that Foxa2 is required for execution of the hepatic gluconeogenic program by integrating the transcriptional response of the hepatocyte to hormonal stimulation.

  12. [Study on antagonistic effect of liangxue huayu recipe on endoplasmic reticulum stress-induced L02 hepatocyte apoptosis and its mechanism].

    PubMed

    Jiang, Ze-Qun; Yao, Zhi-Hua; Deng, Zheng-Ting; Jiang, Xin-Chao; Yan, Xiao-Jing; Chen, Wei-Ping

    2013-10-01

    Endoplasmic reticulum stress (ERS) is a new pathway inducing cell apoptosis that has been discovered in recent years. This study focused on the protective effect of Liangxue Huayu recipe (LHR) on tumor necrosis factor-alpha (TNF-alpha) and D-GalN-induced hepatocyte apoptosis. It found that TNF-alpha and D-GalN could obviously inhibit hepatocyte proliferation, induce cell apoptosis, and significantly increase free calcium ions in cytoplasms, as well as protein expressions of ERS apoptosis-related signal molecules phosphorylated PERK, phosphorylated elF2alpha, cleaved Caspase-12, GRP78 and CHOP. After the administration of LHR of different concentrations, compared with the TNF-alpha/GalN injury group, LHR could significantly alleviated L02 hepatocyte proliferation, decreased cell apoptosis, inhibited growth of intracytoplasmic free calcium content, and gradually reduced the protein expressions of phosphorylated PERK, phosphorylated elF2alpha, cleaved Caspase-12, GRP78 and CHOP. These findings indicated that LHR has the inhibitory effect on TNF-alpha and D-GalN-induced hepatocyte apoptosis. Its mechanism may be related to down-regulation of ERS apoptosis-related signal molecules phosphorylated PERK, phosphorylated elF2alpha, cleaved Caspase-12, GRP78 and CHOP that maintain calcium homeostasis in endoplasmic reticulum.

  13. Autocrine production of TGF-{beta} confers resistance to apoptosis after an epithelial-mesenchymal transition process in hepatocytes: Role of EGF receptor ligands

    SciTech Connect

    Castillo, Gaelle del; Murillo, Miguel M.; Bertran, Esther; Sanchez, Aranzazu; Fabregat, Isabel . E-mail: ifabregat@iro.es

    2006-09-10

    Transforming growth factor-beta (TGF-{beta}) induces apoptosis in fetal rat hepatocytes. However, a subpopulation of these cells survives, concomitant with changes in phenotype, reminiscent of an epithelial-mesenchymal transition (EMT). We have previously suggested that EMT might confer cell resistance to apoptosis (Valdes et al., Mol. Cancer Res., 1: 68-78, 2002). However, the molecular mechanisms responsible for this resistance are not explored yet. In this work, we have isolated and subcultured the population of hepatocytes that suffered the EMT process and are resistant to apoptosis (TGF-{beta}-treated fetal hepatocytes: T{beta}T-FH). We prove that they secrete mitogenic and survival factors, as analyzed by the proliferative and survival capacity of conditioned medium. Inhibition of the epidermal growth factor receptor (EGFR) sensitizes T{beta}T-FH to die after serum withdrawal. T{beta}T-FH expresses high levels of transforming growth factor-alpha (TGF-{alpha}) and heparin-binding EGF-like growth factor (HB-EGF) and shows constitutive activation of the EGFR pathway. A blocking anti-TGF-{alpha} antibody restores the capacity of cells to die. TGF-{beta}, which is expressed by T{beta}T-FH, mediates up-regulation of TGF-{alpha} and HB-EGF expression in those cells. In summary, results suggest that an autocrine loop of TGF-{beta} confers resistance to apoptosis after an EMT process in hepatocytes, through the increase in the expression of EGFR ligands.

  14. [An experimental study on the protective effect of hepatocyte growth factor (HGF) for the primary cultured hepatocytes obtained from iron-loaded rats].

    PubMed

    Yoshida, J

    1995-01-01

    Pathological iron deposition in liver is often found in various liver diseases. The deposited iron is thought to be one of the most important factor of liver cell injury, not only in hemochromotosis but also in cirrhosis following hepatitis virus B or C infection. To investigate the influence of the deposited iron on damage and regeneration of hepatocyte, primary cultured hepatocytes obtained from carbonyl iron-loaded rats were treated with carbon tetrachloride (CCl4) in the presence or absence of hepatocyte growth factor (HGF). Although the section of liver from carbonyl iron-loaded rats showed no necrosis and fibrosis, iron-loaded hepatocytes contained about twofold more iron than control. The damage of iron-loaded hepatocytes induced by CCl4 was more serious than that of control, and HGF decreased this injury only in iron-loaded hepatocytes but not in control. There is no difference in DNA synthesis stimulated by HGF between iron-loaded hepatocytes and control. These findings suggest that the pathological iron deposition induces the fragility of hepatocyte and that cytoprotective effect of HGF is induced by this pathological iron.

  15. Liver tissue engineering utilizing hepatocytes propagated in mouse livers in vivo.

    PubMed

    Ohashi, Kazuo; Tatsumi, Kohei; Tateno, Chise; Kataoka, Miho; Utoh, Rie; Yoshizato, Katsutoshi; Okano, Teruo

    2012-01-01

    Recent advances in tissue engineering technologies have highlighted the ability to create functional liver systems using isolated hepatocytes in vivo. Considering the serious shortage of donor livers that can be used for hepatocyte isolation, it has remained imperative to establish a hepatocyte propagation protocol to provide highly efficient cell recovery allowing for subsequent tissue engineering procedures. Donor primary hepatocytes were isolated from human α-1 antitrypsin (hA1AT) transgenic mice and were transplanted into the recipient liver of urokinase-type plasminogen activator-severe combined immunodeficiency (uPA/SCID) mice. Transplanted donor hepatocytes actively proliferated within the recipient liver of the uPA/SCID mice. At week 8 or later, full repopulation of the uPA/SCID livers with the transplanted hA1AT hepatocytes were confirmed by blood examination and histological assessment. Proliferated hA1AT hepatocytes were recovered from the recipient uPA/SCID mice, and we generated hepatocyte sheets using these recovered hepatocytes for subsequent transplantation into the subcutaneous space of mice. Stable persistency of the subcutaneously engineered liver tissues was confirmed for up to 90 days, which was the length of our present study. These new data demonstrate the feasibility in propagating murine hepatocytes prior to the development of hepatic cells and bioengineered liver systems. The ability to regenerate and expand hepatocytes has potential clinical value whereby procurement of small amounts of tissue could be expanded to sufficient quantities prior to their use in hepatocyte transplantation or other hepatocyte-based therapies.

  16. Insulin-induced CARM1 upregulation facilitates hepatocyte proliferation

    SciTech Connect

    Yeom, Chul-gon; Kim, Dong-il; Park, Min-jung; Choi, Joo-hee; Jeong, Jieun; Wi, Anjin; Park, Whoashig; Han, Ho-jae; Park, Soo-hyun

    2015-06-05

    Previously, we reported that CARM1 undergoes ubiquitination-dependent degradation in renal podocytes. It was also reported that CARM1 is necessary for fasting-induced hepatic gluconeogenesis. Based on these reports, we hypothesized that treatment with insulin, a hormone typically present under the ‘fed’ condition, would inhibit gluconeogenesis via CARM1 degradation. HepG2 cells, AML-12 cells, and rat primary hepatocytes were treated with insulin to confirm CARM1 downregulation. Surprisingly, insulin treatment increased CARM1 expression in all cell types examined. Furthermore, treatment with insulin increased histone 3 methylation at arginine 17 and 26 in HepG2 cells. To elucidate the role of insulin-induced CARM1 upregulation, the HA-CARM1 plasmid was transfected into HepG2 cells. CARM1 overexpression did not increase the expression of lipogenic proteins generally increased by insulin signaling. Moreover, CARM1 knockdown did not influence insulin sensitivity. Insulin is known to facilitate hepatic proliferation. Like insulin, CARM1 overexpression increased CDK2 and CDK4 expression. In addition, CARM1 knockdown reduced the number of insulin-induced G2/M phase cells. Moreover, GFP-CARM1 overexpression increased the number of G2/M phase cells. Based on these results, we concluded that insulin-induced CARM1 upregulation facilitates hepatocyte proliferation. These observations indicate that CARM1 plays an important role in liver pathophysiology. - Highlights: • Insulin treatment increases CARM1 expression in hepatocytes. • CARM1 overexpression does not increase the expression of lipogenic proteins. • CARM1 knockdown does not influence insulin sensitivity. • Insulin-induced CARM1 upregulation facilitates hepatocyte proliferation.

  17. ER stress induces NLRP3 inflammasome activation and hepatocyte death

    PubMed Central

    Lebeaupin, C; Proics, E; de Bieville, C H D; Rousseau, D; Bonnafous, S; Patouraux, S; Adam, G; Lavallard, V J; Rovere, C; Le Thuc, O; Saint-Paul, M C; Anty, R; Schneck, A S; Iannelli, A; Gugenheim, J; Tran, A; Gual, P; Bailly-Maitre, B

    2015-01-01

    The incidence of chronic liver disease is constantly increasing, owing to the obesity epidemic. However, the causes and mechanisms of inflammation-mediated liver damage remain poorly understood. Endoplasmic reticulum (ER) stress is an initiator of cell death and inflammatory mechanisms. Although obesity induces ER stress, the interplay between hepatic ER stress, NLRP3 inflammasome activation and hepatocyte death signaling has not yet been explored during the etiology of chronic liver diseases. Steatosis is a common disorder affecting obese patients; moreover, 25% of these patients develop steatohepatitis with an inherent risk for progression to hepatocarcinoma. Increased plasma LPS levels have been detected in the serum of patients with steatohepatitis. We hypothesized that, as a consequence of increased plasma LPS, ER stress could be induced and lead to NLRP3 inflammasome activation and hepatocyte death associated with steatohepatitis progression. In livers from obese mice, administration of LPS or tunicamycin results in IRE1α and PERK activation, leading to the overexpression of CHOP. This, in turn, activates the NLRP3 inflammasome, subsequently initiating hepatocyte pyroptosis (caspase-1, -11, interleukin-1β secretion) and apoptosis (caspase-3, BH3-only proteins). In contrast, the LPS challenge is blocked by the ER stress inhibitor TUDCA, resulting in: CHOP downregulation, reduced caspase-1, caspase-11, caspase-3 activities, lowered interleukin-1β secretion and rescue from cell death. The central role of CHOP in mediating the activation of proinflammatory caspases and cell death was characterized by performing knockdown experiments in primary mouse hepatocytes. Finally, the analysis of human steatohepatitis liver biopsies showed a correlation between the upregulation of inflammasome and ER stress markers, as well as liver injury. We demonstrate here that ER stress leads to hepatic NLRP3 inflammasome pyroptotic death, thus contributing as a novel mechanism of

  18. Bioactivation of fluorotelomer alcohols in isolated rat hepatocytes.

    PubMed

    Martin, Jonathan W; Chan, Katie; Mabury, Scott A; O'Brien, Peter J

    2009-02-12

    Fluorotelomer alcohols (FTOHs; C(x)F(2x+1)C(2)H(4)OH) are intermediates in the production of specialty surfactants and stain-repellent polymers. The magnitude and pathways of human exposure to FTOHs are not understood, but FTOHs are present in ambient air and house dust, and FTOH-derivatives are used in food-contact applications. Previously, electrophilic FTOH biotransformation products were detected in rat hepatocytes, and liver lesions were found in FTOH exposed rodents. To begin elucidating the mechanism(s) of action, freshly isolated rat hepatocytes were incubated with FTOHs, or FTOH biotransformation products, and toxicity was followed in the presence or absence of carbonyl scavengers and metabolic enzyme modulators. The LC(50) depended on perfluorinated chain length, with the shortest (4:2 FTOH; x=4) and longest (8:2 FTOH; x=8) FTOHs tested being more toxic than the medium chain length FTOH (6:2 FTOH; x=6); a structure-toxicity relationship that is consistent with that for 2-alkenals. For hepatocytes treated with 8:2 FTOH, cytotoxicity corresponded to depletion of glutathione (GSH), increased protein carbonylation, and lipid peroxidation. Aminobenzotriazole, a P450 inhibitor, diminished cytotoxicity for all FTOHs tested, and decreased protein carbonylation and lipid peroxidation for 8:2 FTOH, indicating that a biotransformation product was responsible for FTOH cytotoxicity. Preincubation of hepatocytes with hydralazine or aminoguanidine decreased the cytotoxicity of 8:2 FTOH, suggesting that reactive aldehyde intermediates contributed to the cytotoxicity. A GSH-reactive alpha/beta-unsaturated acid metabolite was also more toxic than the corresponding FTOH, and may have contributed to the observed effects. Overall, these results suggested that FTOH toxicity was related to electrophilic aldehydes or acids through GSH depletion and protein carbonylation. Further research into the nature of protein modification is warranted for these current-use fluorochemicals.

  19. Hepatocyte and Sertoli Cell Aquaporins, Recent Advances and Research Trends

    PubMed Central

    Bernardino, Raquel L.; Marinelli, Raul A.; Maggio, Anna; Gena, Patrizia; Cataldo, Ilaria; Alves, Marco G.; Svelto, Maria; Oliveira, Pedro F.; Calamita, Giuseppe

    2016-01-01

    Aquaporins (AQPs) are proteinaceous channels widespread in nature where they allow facilitated permeation of water and uncharged through cellular membranes. AQPs play a number of important roles in both health and disease. This review focuses on the most recent advances and research trends regarding the expression and modulation, as well as physiological and pathophysiological functions of AQPs in hepatocytes and Sertoli cells (SCs). Besides their involvement in bile formation, hepatocyte AQPs are involved in maintaining energy balance acting in hepatic gluconeogenesis and lipid metabolism, and in critical processes such as ammonia detoxification and mitochondrial output of hydrogen peroxide. Roles are played in clinical disorders including fatty liver disease, diabetes, obesity, cholestasis, hepatic cirrhosis and hepatocarcinoma. In the seminiferous tubules, particularly in SCs, AQPs are also widely expressed and seem to be implicated in the various stages of spermatogenesis. Like in hepatocytes, AQPs may be involved in maintaining energy homeostasis in these cells and have a major role in the metabolic cooperation established in the testicular tissue. Altogether, this information represents the mainstay of current and future investigation in an expanding field. PMID:27409609

  20. Hepatitis C Virus Infects Rhesus Macaque Hepatocytes and Simianized Mice

    PubMed Central

    Scull, Margaret A.; Shi, Chao; de Jong, Ype P.; Gerold, Gisa; Ries, Moritz; von Schaewen, Markus; Donovan, Bridget M.; Labitt, Rachael N.; Horwitz, Joshua A.; Gaska, Jenna M.; Hrebikova, Gabriela; Xiao, Jing W.; Flatley, Brenna; Fung, Canny; Chiriboga, Luis; Walker, Christopher M.; Evans, David T.; Rice, Charles M.; Ploss, Alexander

    2015-01-01

    At least 170 million people are chronically infected with hepatitis C virus (HCV). Due to the narrow host range of HCV and restricted use of chimpanzees, there is currently no suitable animal model for HCV pathogenesis studies or the development of a HCV vaccine. To identify cellular determinants of interspecies transmission and establish a novel immunocompetent model system, we examined the ability of HCV to infect hepatocytes from a small non-human primate, the rhesus macaque (Macaca mulatta). We show that the rhesus orthologs of critical HCV entry factors support viral glycoprotein-dependent virion uptake. Primary hepatocytes from rhesus macaques are also permissive for HCV RNA replication and particle production, which is enhanced when antiviral signaling is suppressed. We demonstrate that this may be due to the diminished capacity of HCV to antagonize MAVS-dependent innate cellular defenses. To test the ability of HCV to establish persistent replication in vivo, we engrafted primary rhesus macaque hepatocytes into immunocompromised xenorecipients. Inoculation of resulting simian liver chimeric mice with either HCV genotype 1a or 2a resulted in HCV serum viremia for up to 10 weeks. Conclusion: Together, these data indicate that rhesus macaques may be a viable model for HCV and implicate host immunity as a potential species-specific barrier to HCV infection. We conclude that suppression of host immunity or further viral adaptation may allow robust HCV infection in rhesus macaques and creation of a new animal model for studies of HCV pathogenesis, lentivirus coinfection and vaccine development. PMID:25820364

  1. Polyethylene glycol protects primary hepatocytes during supercooling preservation.

    PubMed

    Puts, C F; Berendsen, T A; Bruinsma, B G; Ozer, Sinan; Luitje, Martha; Usta, O Berk; Yarmush, M L; Uygun, K

    2015-08-01

    Cold storage (at 4°C) offers a compromise between the benefits and disadvantages of cooling. It allows storage of organs or cells for later use that would otherwise quickly succumb to warm ischemia, but comprises cold ischemia that, when not controlled properly, can result in severe damage as well by both similar and unique mechanisms. We hypothesized that polyethylene glycol (PEG) 35 kDa would ameliorate these injury pathways and improve cold primary hepatocyte preservation. We show that reduction of the storage temperature to below zero by means of supercooling, or subzero non-freezing, together with PEG supplementation increases the viable storage time of primary rat hepatocytes in University of Wisconsin (UW) solution from 1 day to 4 days. We find that the addition of 5% PEG 35 kDa to the storage medium prevents cold-induced lipid peroxidation and maintains hepatocyte viability and functionality during storage. These results suggest that PEG supplementation in combination with supercooling may enable a more optimized cell and organ preservation.

  2. Cellular Redox Modulator, ortho Mn(III) meso-tetrakis(N-n-Hexylpyridinium-2-yl)porphyrin, MnTnHex-2-PyP5+ in the Treatment of Brain Tumors

    PubMed Central

    Keir, Stephen T.; Dewhirst, Mark W.; Kirkpatrick, John P.; Bigner, Darell D.; Batinic-Haberle, Ines

    2012-01-01

    Despite intensive efforts to improve multimodal treatment of brain tumors, survival remains limited. Current therapy consists of a combination of surgery, irradiation and chemotherapy with predisposition to long-term complications. Identifying novel targeted therapies is therefore at the forefront of brain tumor research. This study explores the utility of a manganese porphyrin in a brain tumor model. The compound used is ortho isomer, manganese(III) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin, MnTnHex-2-PyP5+. It is a powerful SOD mimic and peroxynitrite scavenger and a potent modulator of redox-based cellular transcriptional activity, able to suppress excessive immune and inflammatory responses and in turn proliferative pathways. It is further one of the most lipophilic compounds among cationic Mn(III) N-alkylpyridylporphyrins, and thus accumulates predominantly in mitochondria relative to cytosol. In mitochondria, MnTnHex-2-PyP5+ could mimic our key antioxidant system, mitochondrial superoxide dismutase, MnSOD, whose overexpression has been widely shown to suppress tumor growth. Importantly, MnTnHex-2-PyP5+ crosses blood brain barrier in sufficient amounts to demonstrate efficacy in treating CNS injuries. For those reasons we elected to test its effects in inhibiting brain tumor growth. This study is the first report of the antitumor properties of MnTnHex-2-PyP5+ as a single agent in adult and pediatric glioblastoma multiforme (D-54 MG, D-245 MG, D-256 MG, D-456 MG) and pediatric medulloblastoma (D-341 MED), and is the first case where a redox-able metal complex has been used in glioma therapy. When given subcutaneously to mice bearing subcutaneous and intracranial xenografts, MnTnHex-2-PyP5+ caused a significant (P ≤ 0.001) growth delay in D-245 MG, D-256 MG, D-341 MED, and D-456 MG tumors. Growth delay for mice bearing subcutaneous xenografts ranged from 3 days in D-54 MG to 34 days in D-341 MED. With mice bearing intracranial xenografts, MnTnHex-2-PyP5

  3. Hepatocyte-targeted HFE and TFR2 control hepcidin expression in mice.

    PubMed

    Gao, Junwei; Chen, Juxing; De Domenico, Ivana; Koeller, David M; Harding, Cary O; Fleming, Robert E; Koeberl, Dwight D; Enns, Caroline A

    2010-04-22

    Hereditary hemochromatosis is caused by mutations in the hereditary hemochromatosis protein (HFE), transferrin-receptor 2 (TfR2), hemojuvelin, hepcidin, or ferroportin genes. Hepcidin is a key iron regulator, which is secreted by the liver, and decreases serum iron levels by causing the down-regulation of the iron transporter, ferroportin. Mutations in either HFE or TfR2 lower hepcidin levels, implying that both HFE and TfR2 are necessary for regulation of hepcidin expression. In this study, we used a recombinant adeno-associated virus, AAV2/8, for hepatocyte-specific expression of either Hfe or Tfr2 in mice. Expression of Hfe in Hfe-null mice both increased Hfe and hepcidin mRNA and lowered hepatic iron and Tf saturation. Expression of Tfr2 in Tfr2-deficient mice had a similar effect, whereas expression of Hfe in Tfr2-deficient mice or of Tfr2 in Hfe-null mice had no effect on liver or serum iron levels. Expression of Hfe in wild-type mice increased hepcidin mRNA and lowered iron levels. In contrast, expression of Tfr2 had no effect on wild-type mice. These findings suggest that Hfe is limiting in formation of the Hfe/Tfr2 complex that regulates hepcidin expression. In addition, these studies show that the use of recombinant AAV vector to deliver genes is a promising approach for studying physiologic consequences of protein complexes.

  4. Hepatocyte-targeted HFE and TFR2 control hepcidin expression in mice

    PubMed Central

    Gao, Junwei; Chen, Juxing; De Domenico, Ivana; Koeller, David M.; Harding, Cary O.; Fleming, Robert E.; Koeberl, Dwight D.

    2010-01-01

    Hereditary hemochromatosis is caused by mutations in the hereditary hemochromatosis protein (HFE), transferrin-receptor 2 (TfR2), hemojuvelin, hepcidin, or ferroportin genes. Hepcidin is a key iron regulator, which is secreted by the liver, and decreases serum iron levels by causing the down-regulation of the iron transporter, ferroportin. Mutations in either HFE or TfR2 lower hepcidin levels, implying that both HFE and TfR2 are necessary for regulation of hepcidin expression. In this study, we used a recombinant adeno-associated virus, AAV2/8, for hepatocyte-specific expression of either Hfe or Tfr2 in mice. Expression of Hfe in Hfe-null mice both increased Hfe and hepcidin mRNA and lowered hepatic iron and Tf saturation. Expression of Tfr2 in Tfr2-deficient mice had a similar effect, whereas expression of Hfe in Tfr2-deficient mice or of Tfr2 in Hfe-null mice had no effect on liver or serum iron levels. Expression of Hfe in wild-type mice increased hepcidin mRNA and lowered iron levels. In contrast, expression of Tfr2 had no effect on wild-type mice. These findings suggest that Hfe is limiting in formation of the Hfe/Tfr2 complex that regulates hepcidin expression. In addition, these studies show that the use of recombinant AAV vector to deliver genes is a promising approach for studying physiologic consequences of protein complexes. PMID:20177050

  5. Chemokines induce axon outgrowth downstream of Hepatocyte Growth Factor and TCF/β-catenin signaling

    PubMed Central

    Bhardwaj, Deepshikha; Náger, Mireia; Camats, Judith; David, Monica; Benguria, Alberto; Dopazo, Ana; Cantí, Carles; Herreros, Judit

    2013-01-01

    Axon morphogenesis is a complex process regulated by a variety of secreted molecules, including morphogens and growth factors, resulting in the establishment of the neuronal circuitry. Our previous work demonstrated that growth factors [Neurotrophins (NT) and Hepatocyte Growth Factor (HGF)] signal through β-catenin during axon morphogenesis. HGF signaling promotes axon outgrowth and branching by inducing β-catenin phosphorylation at Y142 and transcriptional regulation of T-Cell Factor (TCF) target genes. Here, we asked which genes are regulated by HGF signaling during axon morphogenesis. An array screening indicated that HGF signaling elevates the expression of chemokines of the CC and CXC families. In line with this, CCL7, CCL20, and CXCL2 significantly increase axon outgrowth in hippocampal neurons. Experiments using blocking antibodies and chemokine receptor antagonists demonstrate that chemokines act downstream of HGF signaling during axon morphogenesis. In addition, qPCR data demonstrates that CXCL2 and CCL5 expression is stimulated by HGF through Met/b-catenin/TCF pathway. These results identify CC family members and CXCL2 chemokines as novel regulators of axon morphogenesis downstream of HGF signaling. PMID:23641195

  6. Suppression of Hepatic Cyp1a2 by Total Ginsenosides in Lipopolysaccharide-Treated Mice and Primary Mouse Hepatocytes.

    PubMed

    Sun, Haiyan; Yan, Yijing; Xu, Chenshu; Wan, Hongxia; Liu, Dong

    2016-03-23

    The roots of Panax ginseng (ginseng) have been extensively used in traditional Chinese medicine. However, herb-drug interactions between ginseng and other co-administered drugs are not fully understood concerning the effect of ginseng on drug metabolism and clearance. The current study aimed to elucidate the effect of total ginsenosides, a typical ginseng extract, on the regulation of Cyp1a2, a key enzyme to regulate drug metabolism under the normal and inflammatory conditions in mice. Female C57BL/6J mice treated with vehicle and lipopolysaccharide (LPS) were intragastrically administered ginseng extract for 7 days before hepatic P450 expression was analyzed. Primary mouse hepatocytes were also employed to further explore the effects of total ginsenosides on Cyp1a2 expression. The results showed that total ginsenosides in P. ginseng extract exhibited a concentration-dependent suppression on Cyp1a2 mRNA and protein level in both mice and primary mouse hepatocytes. Notably, the inhibitory effects of total ginsenosides on Cyp1a2 mRNA and protein expression were further enhanced following LPS treatment. Therefore, future research is warranted to investigate the role of ginsenosides in the regulation of hepatic CYP450s. Moreover, consumption of ginseng as food or supplement should be monitored for patients on combinational therapy, especially those with inflammatory diseases.

  7. trans-Dichloridobis{dicyclo­hex­yl[4-(dimethyl­amino)­phen­yl]phosphane-κP}platinum(II) dichloro­methane disolvate

    PubMed Central

    Davis, Wade L.; Meijboom, Reinout

    2012-01-01

    In the title complex, trans-[PtCl2{P(C6H11)2(4-Me2NC6H4)}2]·2CH2Cl2, the PtII atom is located on an inversion centre, resulting in a trans-square-planar geometry. Important geometric parameters are the Pt—P and Pt—Cl bond lengths of 2.3258 (6) and 2.3106 (6) Å, respectively, and the P—Pt—Cl angles of 89.64 (2) and 90.36 (2)°. The effective cone angle for the dicyclo­hex­yl[4-(dimethyl­amino)­phen­yl]phosphane unit was calculated to be 164°. The compound crystallizes with two dichloro­methane solvent mol­ecules; one of which is severely disordered and was treated using the SQUEEZE routine in PLATON [Spek (2009 ▶). Acta Cryst. D65, 148–155]. PMID:23468763

  8. Species-specific toxicity of troglitazone on rats and human by gel entrapped hepatocytes

    SciTech Connect

    Shen, Chong; Meng, Qin; Zhang, Guoliang

    2012-01-01

    Troglitazone, despite passing preclinical trials on animals, was shortly withdrawn from market due to its severe hepatotoxicity in clinic. As rat hepatocyte monolayer consistently showed sensitive troglitazone toxicity as human hepatocyte monolayer in contrast to the species-specific toxicity in vivo, this paper utilized both hepatocytes in three-dimensional culture of gel entrapment to reflect the species difference on hepatotoxicity. Rat hepatocytes in gel entrapment did not show obvious cellular damage even under a long-term exposure for 21 days while gel entrapped human hepatocytes significantly displayed oxidative stress, steatosis, mitochondrial damage and cell death at a short exposure for 4 days. As a result, the detected species-specific toxicity of troglitazone between gel entrapped rat and human hepatocytes consisted well with the situation in vivo but was in a sharp contrast to the performance of two hepatocytes by monolayer culture. Such contradictory toxicity of rat hepatocytes between monolayer and gel entrapment culture could be explained by the fact that troglitazone was cleared more rapidly in gel entrapment than in monolayer culture. Similarly, the differential clearance of troglitazone in rat and human might also explain its species-specific toxicity. Therefore, gel entrapment of hepatocytes might serve as a platform for evaluation of drug toxicity at early stage of drug development by reducing costs, increasing the likelihood of clinical success and limiting human exposure to unsafe drugs. -- Highlights: ► Species-specific toxicity of troglitazone reflected by rat/human hepatocytes ► 3D hepatocytes in 21 days’ long-term culture used for drug hepatotoxicity ► Oversensitive toxicity in hepatocyte monolayer by slow troglitazone clearance.

  9. Cytokine and chemokine expression associated with steatohepatitis and hepatocyte proliferation in rats fed ethanol via total enteral nutrition.

    PubMed

    Ronis, Martin J J; Butura, Angelica; Korourian, Soheila; Shankar, Kartik; Simpson, Pippa; Badeaux, Jamie; Albano, Emanuele; Ingelman-Sundberg, Magnus; Badger, Thomas M

    2008-03-01

    To determine the temporal relationship between alcohol-induced changes in cytokines and chemokines, development of liver pathology and stimulation of hepatocyte proliferation, male Sprague-Dawley rats were intragastrically fed low carbohydrate-containing ethanol (EtOH) diets via total enteral nutrition (TEN) for up to 49 d. Induction of EtOH metabolism and appearance of steatosis preceded development of oxidative stress, inflammation, and cell death. A transitory peak of tumor necrosis factor (TNFalpha) and interferon gamma (IFN gamma) was observed at 14 d followed by reduced expression of TNFalpha, IFN gamma and another Th1 cytokine IL-12 accompanied by reduced expression of the Th1 regulators T-bet and STAT4. After 35-49 d of EtOH, at a time when hepatocyte proliferation was stimulated, IL-12 returned to control values and a second peak of TNFalpha occurred. The Th2 cytokine IL-4 remained suppressed throughout the study and was accompanied by reductions in the Th2 regulator GATA3. There was no temporal effect of EtOH on expression of IL-6 or TGFbeta. IL-5 and IL-13 mRNA were undetectable. Chemokine CXCL-2 expression increased progressively up to 35 d and preceded the appearance of inflammatory infiltrates. These data suggest that steatosis, increased ethanol metabolism, a transient induction of the innate immune response and suppression of Th2 responses were acute consequences of ethanol treatment and were followed by suppression of Th1 responses. However, the majority of necrosis, apoptosis and a late peak of TNFalpha only occurred after 6-7 weeks of ethanol, coincided with the appearance of inflammatory infiltrates and were associated with stimulation of hepatocyte proliferation.

  10. AMPK Activation Prevents and Reverses Drug-Induced Mitochondrial and Hepatocyte Injury by Promoting Mitochondrial Fusion and Function

    PubMed Central

    Taniane, Caitlin; Farrell, Geoffrey; Arias, Irwin M.; Lippincott-Schwartz, Jennifer; Fu, Dong

    2016-01-01

    Mitochondrial damage is the major factor underlying drug-induced liver disease but whether conditions that thwart mitochondrial injury can prevent or reverse drug-induced liver damage is unclear. A key molecule regulating mitochondria quality control is AMP activated kinase (AMPK). When activated, AMPK causes mitochondria to elongate/fuse and proliferate, with mitochondria now producing more ATP and less reactive oxygen species. Autophagy is also triggered, a process capable of removing damaged/defective mitochondria. To explore whether AMPK activation could potentially prevent or reverse the effects of drug-induced mitochondrial and hepatocellular damage, we added an AMPK activator to collagen sandwich cultures of rat and human hepatocytes exposed to the hepatotoxic drugs, acetaminophen or diclofenac. In the absence of AMPK activation, the drugs caused hepatocytes to lose polarized morphology and have significantly decreased ATP levels and viability. At the subcellular level, mitochondria underwent fragmentation and had decreased membrane potential due to decreased expression of the mitochondrial fusion proteins Mfn1, 2 and/or Opa1. Adding AICAR, a specific AMPK activator, at the time of drug exposure prevented and reversed these effects. The mitochondria became highly fused and ATP production increased, and hepatocytes maintained polarized morphology. In exploring the mechanism responsible for this preventive and reversal effect, we found that AMPK activation prevented drug-mediated decreases in Mfn1, 2 and Opa1. AMPK activation also stimulated autophagy/mitophagy, most significantly in acetaminophen-treated cells. These results suggest that activation of AMPK prevents/reverses drug-induced mitochondrial and hepatocellular damage through regulation of mitochondrial fusion and autophagy, making it a potentially valuable approach for treatment of drug-induced liver injury. PMID:27792760

  11. Induction of cytochrome P4501A by highly purified hexachlorobenzene in primary cultures of ring-necked pheasant and Japanese quail embryo hepatocytes.

    PubMed

    Mundy, Lukas J; Crump, Doug; Jones, Stephanie P; Konstantinov, Alex; Utley, Fiona; Potter, David; Kennedy, Sean W

    2012-04-01

    Primary cultures of ring-necked pheasant (Phasianus colchicus) and Japanese quail (Coturnix japonica) embryo hepatocytes were used to compare the potencies of highly purified hexachlorobenzne (HCB-P), reagent-grade HCB (RG-HCB) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as inducers of ethoxyresorufin O-deethylase (EROD) activity, cytochrome P4501A (CYP1A4) messenger ribonucleic acid (mRNA) and CYP1A5 mRNA. HCB-P, RG-HCB and TCDD all induced EROD activity and up-regulated CYP1A4 and CYP1A5 mRNA. Induction was not caused by contamination of HCB with polychlorinated dibenzo-p-dioxins, dibenzofurans or biphenyls. Based upon a comparison of the EC(50) and EC(threshold) values for EROD and CYP1A4/5 concentration-response curves, the potency of HCB relative to TCDD was 0.001 in ring-necked pheasant and 0.01 in Japanese quail embryo hepatocytes. Differences in species sensitivity to HCB were found to be mainly dictated by differences in species sensitivity to TCDD rather than differences in the absolute potency of HCB. Consequently, ring-necked pheasant and Japanese quail embryo hepatocytes were found to be equally sensitive to HCB exposure. Species sensitivity comparisons were also made with chicken (Gallus gallus domesticus) and revealed that chicken embryo hepatocytes were less responsive to EROD induction (lower maximal response) by HCB compared to the embryo hepatocytes of pheasant and quail.

  12. Nicotinamide ameliorates palmitate-induced ER stress in hepatocytes via cAMP/PKA/CREB pathway-dependent Sirt1 upregulation.

    PubMed

    Li, Jiaxin; Dou, Xiaobing; Li, Songtao; Zhang, Ximei; Zeng, Yong; Song, Zhenyuan

    2015-11-01

    Nicotinamide (NAM) is the amide of nicotinic acid and a predominant precursor for NAD(+) biosynthesis via the salvage pathway. Sirt1 is a NAD(+)-dependent deacetylase, playing an important role in regulating cellular functions. Although hepatoprotective effect of NAM has been reported, the underlying mechanism remains elusive. ER stress, induced by saturated fatty acids, in specific palmitate, plays a pathological role in the development of nonalcoholic fatty liver disease. This study aims to determine the effect of NAM on palmitate-induced ER stress in hepatocytes and to elucidate molecular mechanisms behind. Both HepG2 cells and primary mouse hepatocytes were exposed to palmitate (conjugated to BSA at a 2:1 M ratio), NAM, or their combination for different durations. Cellular NAD(+) level, Sirt1 expression/activity, ER stress, as well as cAMP/PKA/CREB pathway activation were determined. NAM increased Sirt1 expression and enzymatic activity, which contributes to the ameliorative effect of NAM on palmitate-triggered ER stress. NAM increased intracellular NAD(+) level in hepatocytes, however, blocking the salvage pathway, a pathway for NAD(+) synthesis from NAM, only partially prevented NAM-induced Sirt1 upregulation while completely prevented NAD+ increase in response to NAM. Further mechanistic investigations revealed that NAM elevated intracellular cAMP level via suppressing PDE activity, leading to downstream PKA and CREB activation. Importantly, cAMP/PKA/CREB pathway blockade abolished not only NAM-induced Sirt1 upregulation, but also its protective effect against ER stress. Our results demonstrate that NAM protects hepatocytes against palmitate-induced ER stress in hepatocytes via upregulating Sirt1. Activation of the cAMP/PKA/CREB pathway plays a key role in NAM-induced Sirt1 upregulation.

  13. A novel experimental strategy to assess the metabolic effects of selective activation of a G(q)-coupled receptor in hepatocytes in vivo.

    PubMed

    Li, Jian Hua; Jain, Shalini; McMillin, Sara M; Cui, Yinghong; Gautam, Dinesh; Sakamoto, Wataru; Lu, Huiyan; Jou, William; McGuinness, Owen P; Gavrilova, Oksana; Wess, Jürgen

    2013-10-01

    Increased hepatic glucose production is a key pathophysiological feature of type 2 diabetes. Like all other cell types, hepatocytes express many G protein-coupled receptors (GPCRs) that are linked to different functional classes of heterotrimeric G proteins. The important physiological functions mediated by G(s)-coupled hepatic glucagon receptors are well-documented. In contrast, little is known about the in vivo physiological roles of hepatocyte GPCRs that are linked to G proteins of the G(q) family. To address this issue, we established a transgenic mouse line (Hep-Rq mice) that expressed a G(q)-linked designer receptor (Rq) in a hepatocyte-selective fashion. Importantly, Rq could no longer bind endogenous ligands but could be selectively activated by a synthetic drug, clozapine-N-oxide. Clozapine-N-oxide treatment of Hep-Rq mice enabled us to determine the metabolic consequences caused by selective activation of a G(q)-coupled GPCR in hepatocytes in vivo. We found that acute Rq activation in vivo led to pronounced increases in blood glucose levels, resulting from increased rates of glycogen breakdown and gluconeogenesis. We also demonstrated that the expression of the V(1b) vasopressin receptor, a G(q)-coupled receptor expressed by hepatocytes, was drastically increased in livers of ob/ob mice, a mouse model of diabetes. Strikingly, treatment of ob/ob mice with a selective V(1b)