Liver Proteomic Response To Hypertriglyceridemia In Human-apolipoprotein C-iii Transgenic Mice At Cellular And Mitochondrial Compartment Levels

Background: Hypertriglyceridemia (HTG) is defined as a triglyceride (TG) plasma level exceeding 150 mg/dl and is tightly associated with atherosclerosis, metabolic syndrome, obesity, diabetes and acute pancreatitis. The present study was undertaken to investigate the mitochondrial, sub-mitochondrial and cellular proteomic impact of hypertriglyceridemia in the hepatocytes of hypertriglyceridemic transgenic mice (overexpressing the human apolipoproteinC-III). Methods. Quantitative proteomics (2D-DIGE) analysis was carried out on both "low-expressor" (LE) and "high- expressor" (HE) mice, respectively exhibiting moderate and severe HTG, to characterize the effect of the TG plasma level on the proteomic response. Results: The mitoproteome analysis has revealed a large-scale phenomenon in transgenic mice, i.e. a general down-regulation of matricial proteins and up-regulation of inner membrane proteins. These data also demonstrate that the magnitude of proteomic changes strongly depends on the TG plasma level. Our different analyses indicate that, in HE mice, the capacity of several metabolic pathways is altered to promote the availability of acetyl-CoA, glycerol-3-phosphate, ATP and NADPH for TG de novo biosynthesis. The up-regulation of several cytosolic ROS detoxifying enzymes has also been observed, suggesting that the cytoplasm of HTG mice is subjected to oxidative stress. Moreover, our results suggest that iron over-accumulation takes place in the cytosol of HE mice hepatocytes and may contribute to enhance oxidative stress and to promote cellular proliferation. Conclusions: These results indicate that the metabolic response to HTG in human apolipoprotein C-III overexpressing mice may support a high TG production rate and that the cytosol of hepatocytes is subjected to an important oxidative stress, probably as a result of FFA over-accumulation, iron overload and enhanced activity of some ROS-producing catabolic enzymes. © 2014 Ehx et al.; licensee BioMed Central Ltd.131Grundy, S.M., Brewer Jr., H.B., Cleeman, J.I., Smith Jr., S.C., Lenfant, C., Definition of Metabolic Syndrome: Report of the National Heart, Lung, and Blood Institute/American Heart Association Conference on Scientific Issues Related to Definition (2004) Circulation, 109 (3), pp. 433-438. , DOI 10.1161/01.CIR.0000111245.75752.C6Reid, A.E., Nonalcoholic steatohepatitis (2001) Gastroenterology, 121 (3), pp. 710-723Toskes, P.P., Hyperlipidemic pancreatitis (1990) Gastroenterol Clin North Am, 19, pp. 783-791Ginsberg, H.N., Ngoc-Anh, L., Goldberg, I.J., Apolipoprotein B metabolism in subjects with deficiency of apolipoproteins CIII and AI. Evidence that apolipoprotein CIII inhibits catabolism of triglyceride-rich lipoproteins by lipoprotein lipase in vivo (1986) Journal of Clinical Investigation, 78 (5), pp. 1287-1295Maeda, N., Li, H., Lee, D., Oliver, P., Quarfordt, S.H., Osada, J., Targeted disruption of the apolipoprotein C-III gene in mice results in hypotriglyceridemia and protection from postprandial hypertriglyceridemia (1994) Journal of Biological Chemistry, 269 (38), pp. 23610-23616Shoulders, C.C., Harry, P.J., Lagrost, L., White, S.E., Shah, N.F., North, J.D., Gilligan, M., Ball, M.J., Variation at the apo AI/CIII/AIV gene complex is associated with elevated plasma levels of apo CIII (1991) Atherosclerosis, 87, pp. 239-247Jong, M.C., Rensen, P.C.N., Dahlmans, V.E.H., Van Der Boom, H., Van Berkel, T.J.C., Havekes, L.M., Apolipoprotein C-III deficiency accelerates triglyceride hydrolysis by lipoprotein lipase in wild-type and apoE knockout mice (2001) Journal of Lipid Research, 42 (10), pp. 1578-1585Wang, C.-S., McConathy, W., Kloer, H.U., Alaupovic, P., Modulation of lipoprotein lipase activity by apolipoproteins. Effect of apolipoprotein C-III (1985) Journal of Clinical Investigation, 75 (2), pp. 384-390Mann, C.J., Troussard, A.A., Yen, F.T., Hannouche, N., Najib, J., Fruchart, J.-C., Lotteau, V., Bihain, B.E., Inhibitory effects of specific apolipoprotein C-III isoforms on the binding of triglyceride-rich lipoproteins to the lipolysis-stimulated receptor (1997) Journal of Biological Chemistry, 272 (50), pp. 31348-31354. , DOI 10.1074/jbc.272.50.31348Windler, E., Havel, R.J., Inhibitory effects of C apolipoproteins from rats and humans on the uptake of triglyceride-rich lipoproteins and their remnants by the perfused rat liver (1985) Journal of Lipid Research, 26 (5), pp. 556-565Ito, Y., Azrolan, N., O'Connell, A., Walsh, A., Breslow, J.L., Hypertriglyceridemia as a result of human apo CIII gene expression in transgenic mice (1990) Science, 249, pp. 790-793Aalto-Setala, K., Fisher, E.A., Chen, X., Chajek-Shaul, T., Hayek, T., Zechner, R., Walsh, A., Breslow, J.L., Mechanism of hypertriglyceridemia in human apolipoprotein (apo) CIII transgenic mice. Diminished very low density lipoprotein fractional catabolic rate associated with increased apo CIII and reduced apo e on the particles (1992) J Clin Invest, 90, pp. 1889-1900Reaven, G.M., Mondon, C.E., Chen, Y.-D.I., Breslow, J.L., Hypertriglyceridemic mice transgenic for the human apolipoprotein C-III gene are neither insulin resistant nor hyperinsulinemic (1994) Journal of Lipid Research, 35 (5), pp. 820-824Alberici, L.C., Oliveira, H.C.F., Patricio, P.R., Kowaltowski, A.J., Vercesi, A.E., Hyperlipidemic Mice Present Enhanced Catabolism and Higher Mitochondrial ATP-Sensitive K+ Channel Activity (2006) Gastroenterology, 131 (4), pp. 1228-1234. , DOI 10.1053/j.gastro.2006.07.021, PII S0016508506016647Salerno, A.G., Silva, T.R., Amaral, M.E.C., Alberici, L.C., Bonfleur, M.L., Patricio, P.R., Francesconi, E.P.M.S., Oliveira, H.C.F., Overexpression of apolipoprotein CIII increases and CETP reverses diet-induced obesity in transgenic mice (2007) International Journal of Obesity, 31 (10), pp. 1586-1595. , DOI 10.1038/sj.ijo.0803646, PII 0803646Amaral, M.E.C., Oliveira, H.C.F., Carneiro, E.M., Delghingaro-Augusto, V., Vieira, E.C., Berti, J.A., Boschero, A.C., Plasma glucose regulation and insulin secretion in hypertriglyceridemic mice (2002) Hormone and Metabolic Research, 34 (1), pp. 21-26. , DOI 10.1055/s-2002-19962Alberici, L.C., Vercesi, A.E., Oliveira, H.C., Mitochondrial energy metabolism and redox responses to hypertriglyceridemia (2011) J Bioenerg Biomembr, 43, pp. 19-23Alberici, L.C., Oliveira, H.C.F., Bighetti, E.J.B., De Faria, E.C., Degaspari, G.R., Souza, C.T., Vercesi, A.E., Hypertriglyceridemia Increases Mitochondrial Resting Respiration and Susceptibility to Permeability Transition (2003) Journal of Bioenergetics and Biomembranes, 35 (5), pp. 451-457. , DOI 10.1023/A:1027343915452Garlid, K.D., Paucek, P., Mitochondrial potassium transport: The K(+) cycle (2003) Biochim Biophys Acta, 1606, pp. 23-41Alberici, L.C., Oliveira, H.C., Paim, B.A., Mantello, C.C., Augusto, A.C., Zecchin, K.G., Gurgueira, S.A., Vercesi, A.E., Mitochondrial ATP-sensitive K(+) channels as redox signals to liver mitochondria in response to hypertriglyceridemia (2009) Free Radic Biol Med, 47, pp. 1432-1439Mathy, G., Sluse, F.E., Mitochondrial comparative proteomics: Strengths and pitfalls (2008) Biochim Biophys Acta, 1777, pp. 1072-1077Douette, P., Navet, R., Gerkens, P., De Pauw, E., Leprince, P., Sluse-Goffart, C., Sluse, F.E., Steatosis-induced proteomic changes in liver mitochondria evidenced by two-dimensional differential in-gel electrophoresis (2005) Journal of Proteome Research, 4 (6), pp. 2024-2031. , DOI 10.1021/pr050187zMarouga, R., David, S., Hawkins, E., The development of the DIGE system: 2D fluorescence difference gel analysis technology (2005) Analytical and Bioanalytical Chemistry, 382 (3), pp. 669-678. , DOI 10.1007/s00216-005-3126-3Sue, G.R., Ho, Z.C., Kim, K., Peroxiredoxins: A historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling (2005) Free Radical Biology and Medicine, 38 (12), pp. 1543-1552. , DOI 10.1016/j.freeradbiomed.2005.02.026, PII S0891584905000985Hayes, J.D., Flanagan, J.U., Jowsey, I.R., Glutathione transferases (2005) Annual Review of Pharmacology and Toxicology, 45, pp. 51-88. , DOI 10.1146/annurev.pharmtox.45.120403.095857Raisanen, S.R., Lehenkari, P., Tasanen, M., Rahkila, P., Harkonen, P.L., Vaananen, H.K., Carbonic anhydrase III protects cells from hydrogen peroxide-induced apoptosis (1999) FASEB Journal, 13 (3), pp. 513-522Zheng, J., Li, Y., Yang, J., Liu, Q., Shi, M., Zhang, R., Shi, H., Liu, W., NDRG2 inhibits hepatocellular carcinoma adhesion, migration and invasion by regulating CD24 expression (2011) BMC Cancer, 11 (251), pp. 251-259Lee, H.Y., Birkenfeld, A.L., Jornayvaz, F.R., Jurczak, M.J., Kanda, S., Popov, V., Frederick, D.W., Shulman, G.I., Apolipoprotein CIII overexpressing mice are predisposed to diet-induced hepatic steatosis and hepatic insulin resistance (2011) Hepatology, 54, pp. 1650-1660Mathy, G., Navet, R., Gerkens, P., Leprince, P., De Pauw, E., Sluse-Goffart, C.M., Sluse, F.E., Douette, P., Saccharomyces cerevisiae mitoproteome plasticity in response to recombinant alternative ubiquinol oxidase (2006) Journal of Proteome Research, 5 (2), pp. 339-348. , DOI 10.1021/pr050346eKnowles, M.R., Cervino, S., Skynner, H.A., Hunt, S.P., De Felipe, C., Salim, K., Meneses-Lorente, G., Guest, P.C., Multiplex proteomic analysis by two-dimensional differential in-gel electrophoresis (2003) Proteomics, 3 (7), pp. 1162-1171. , DOI 10.1002/pmic.200300437Cardoso, A.R., Queliconi, B.B., Kowaltowski, A.J., Mitochondrial ion transport pathways: Role in metabolic diseases (2010) Biochim Biophys Acta, 1797, pp. 832-838Bonnard, C., Durand, A., Peyrol, S., Chanseaume, E., Chauvin, M.-A., Morio, B., Vidal, H., Rieusset, J., Mitochondrial dysfunction results from oxidative stress in the skeletal muscle of diet-induced insulin-resistant mice (2008) Journal of Clinical Investigation, 118 (2), pp. 789-800. , http://www.jci.org/articles/view/32601/pdf, DOI 10.1172/JCI32601Haynes, C.M., Fiorese, C.J., Lin, Y.F., Evaluating and responding to mitochondrial dysfunction: The mitochondrial unfolded-protein response and beyond (2013) Trends Cell Biol, 23, pp. 311-318MacGarvey, N.C., Suliman, H.B., Bartz, R.R., Fu, P., Withers, C.M., Welty-Wolf, K.E., Piantadosi, C.A., Activation of mitochondrial biogenesis by heme oxygenase-1-mediated NF-E2-related factor-2 induction rescues mice from lethal Staphylococcus aureus sepsis (2012) Am J Respir Crit Care Med, 185, pp. 851-861Piao, C.S., Gao, S., Lee, G.H., Kim Do, S., Park, B.H., Chae, S.W., Chae, H.J., Kim, S.H., Sulforaphane protects ischemic injury of hearts through antioxidant pathway and mitochondrial K(ATP) channels (2010) Pharmacol Res, 61, pp. 342-348Fodor, I.K., Nelson, D.O., Alegria-Hartman, M., Robbins, K., Langlois, R.G., Turteltaub, K.W., Corzett, T.H., McCutchen-Maloney, S.L., Statistical challenges in the analysis of two-dimensional difference gel electrophoresis experiments using DeCyder™ (2005) Bioinformatics, 21 (19), pp. 3733-3740. , DOI 10.1093/bioinformatics/bti612Callister, S.J., Barry, R.C., Adkins, J.N., Johnson, E.T., Qian, W.-J., Webb-Robertson, B.-J.M., Smith, R.D., Lipton, M.S., Normalization approaches for removing systematic biases associated with mass spectrometry and label-free proteomics (2006) Journal of Proteome Research, 5 (2), pp. 277-286. , DOI 10.1021/pr050300lNg, D.S., Xie, C., Maguire, G.F., Zhu, X., Ugwu, F., Lam, E., Connelly, P.W., Hypertriglyceridemia in Lecithin-cholesterol Acyltransferase-deficent Mice Is Associated with Hepatic Overproduction of Triglycerides, Increased Lipogenesis, and Improved Glucose Tolerance (2004) Journal of Biological Chemistry, 279 (9), pp. 7636-7642. , DOI 10.1074/jbc.M309439200Li, L.O., Hu, Y.F., Wang, L., Mitchell, M., Berger, A., Coleman, R.A., Early hepatic insulin resistance in mice: A metabolomics analysis (2010) Mol Endocrinol, 24, pp. 657-666Thomas, A., Stevens, A.P., Klein, M.S., Hellerbrand, C., Dettmer, K., Gronwald, W., Oefner, P.J., Reinders, J., Early changes in the liver-soluble proteome from mice fed a nonalcoholic steatohepatitis inducing diet (2012) Proteomics, 12, pp. 1437-1451Ray, P.D., Huang, B.W., Tsuji, Y., Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling (2012) Cell Signal, 24, pp. 981-990Dansen, T.B., Wirtz, K.W.A., The peroxisome in oxidative stress (2001) IUBMB Life, 51 (4), pp. 223-230. , DOI 10.1080/152165401753311762Leclercq, I.A., Farrell, G.C., Field, J., Bell, D.R., Gonzalez, F.J., Robertson, G.R., CYP2E1 and CYP4A as microsomal catalysts of lipid peroxides in murine nonalcoholic steatohepatitis (2000) Journal of Clinical Investigation, 105 (8), pp. 1067-1075Zhang, Y.K., Wu, K.C., Klaassen, C.D., Genetic activation of Nrf2 protects against fasting-induced oxidative stress in livers of mice (2013) PLoS One, 8, p. 559122Mackenzie, E.L., Iwasaki, K., Tsuji, Y., Intracellular iron transport and storage: From molecular mechanisms to health implications (2008) Antioxidants and Redox Signaling, 10 (6), pp. 997-1030. , DOI 10.1089/ars.2007.1893Dongiovanni, P., Fracanzani, A.L., Fargion, S., Valenti, L., Iron in fatty liver and in the metabolic syndrome: A promising therapeutic target (2011) J Hepatol, 55, pp. 920-932Rouault, T.A., Hentze, M.W., Caughman, S.W., Harford, J.B., Klausner, R.D., Binding of a cytosolic protein to the iron-responsive element of human ferritin messenger RNA (1988) Science, 241, pp. 1207-1210Hentze, M.W., Caughman, S.W., Rouault, T.A., Barriocanal, J.G., Dancis, A., Harford, J.B., Klausner, R.D., Identification of the iron responsive element for the translational regulation of human ferritin mRNA (1987) Science, 238 (4833), pp. 1570-1573Petrak, J., Myslivcova, D., Man, P., Cmejla, R., Cmejlova, J., Vyoral, D., Elleder, M., Vulpe, C.D., Proteomic analysis of hepatic iron overload in mice suggests dysregulation of urea cycle, impairment of fatty acid oxidation, and changes in the methylation cycle (2007) Am J Physiol Gastrointest Liver Physiol, 292, pp. 71490-G1498Krawczyk, M., Bonfrate, L., Portincasa, P., Nonalcoholic fatty liver disease (2010) Best Pract Res Clin Gastroenterol, 24, pp. 695-708Koek, G.H., Liedorp, P.R., Bast, A., The role of oxidative stress in non-alcoholic steatohepatitis (2011) Clin Chim Acta, 412, pp. 1297-1305Caldwell, S.H., De Freitas, L.A., Park, S.H., Moreno, M.L., Redick, J.A., Davis, C.A., Sisson, B.J., Al-Osaimi, A., Intramitochondrial crystalline inclusions in nonalcoholic steatohepatitis (2009) Hepatology, 49, pp. 1888-1895Sickmann, A., Reinders, J., Wagner, Y., Joppich, C., Zahedi, R., Meyer, H.E., Schonfisch, B., Meisinger, C., The proteome of Saccharomyces cerevisiae mitochondria (2003) Proceedings of the National Academy of Sciences of the United States of America, 100 (23), pp. 13207-13212. , DOI 10.1073/pnas.2135385100Sottocasa, G.L., Kuylenstierna, B., Ernster, L., Bergstrand, A., An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study (1967) J Cell Biol, 32, pp. 415-438Hurkman, W.J., Tanaka, C.K., Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis (1986) Plant Physiol, 81, pp. 802-806Shevchenko, An., Wilm, M., Vorm, O., Jensen, O.N., Podtelejnikov, A.V., Neubauer, G., Shevchenko, Al., Mann, M., A strategy for identifying gel-separated proteins in sequence databases by MS alone (1996) Biochemical Society Transactions, 24 (3), pp. 893-89

Azacytidine Enhances Regulatory T-Cells In Vivo and Prevents Experimental Xenogeneic Graft-Versus-Host Disease

Background The demethylating agent 5-azacytidine (AZA) has proven its efficacy as treatment for myelodysplastic syndrome and acute myeloid leukemia. In addition, AZA can demethylate FOXP3 intron 1 (FOXP3i1) leading to the generation of regulatory T cells (Tregs). Objective We investigated the impact of AZA on xenogeneic graft-versus-host disease (xGVHD) in a humanized murine model of transplantation, and described the impact of the drug on human T cells in vivo. Methods In order to induce xGVHD, human peripheral blood mononuclear cells (huPBMC) were administered intravenously in NOD-scid IL-2Rγnull (NSG) mice. Results AZA successfully improved both survival (p<0.0001) and xGVHD scores (p<0.0001). Further, AZA significantly decreased human T-cell proliferation as well as INF-γ and TNF-α serum levels, and reduced the expression of GRANZYME B and PERFORIN 1 by cytotoxic T cells. In addition, AZA administration significantly increased the function, proliferation and frequency of Tregs through demethylation of FOXP3i1 and higher secretion of IL-2 by conventional T cells due to IL2 gene promoter site 1 demethylation. Interestingly, among AZA-treated mice surviving the acute phase of xGVHD, there was an inverse correlation between the presence of Tregs and signs of chronic GVHD. Finally, Tregs harvested from the spleen of AZA-treated mice were suppressive and stable over time since they persisted at high frequency in secondary transplant experiments. Conclusion These findings emphasize a potential role for AZA as prevention or treatment of GVHD

The prosurvival IKK-related kinase IKKϵ integrates LPS and IL17A signaling cascades to promote Wnt-dependent tumor development in the intestine

Constitutive Wnt signaling promotes intestinal cell proliferation, but signals from the tumor microenvironment are also required to support cancer development. The role that signaling proteins play to establish a tumor microenvironment has not been extensively studied. Therefore, we assessed the role of the proinflammatory Ikk-related kinase Ikkϵ in Wnt-driven tumor development. We found that Ikkϵ was activated in intestinal tumors forming upon loss of the tumor suppressor Apc. Genetic ablation of Ikkϵ in b-catenin-driven models of intestinal cancer reduced tumor incidence and consequently extended survival. Mechanistically, we attributed the tumor-promoting effects of Ikkϵ to limited TNF-dependent apoptosis in transformed intestinal epithelial cells. In addition, Ikkϵ was also required for lipopolysaccharide (LPS) and IL17A-induced activation of Akt, Mek1/2, Erk1/2, and Msk1. Accordingly, genes encoding proinflammatory cytokines, chemokines, and anti-microbial peptides were downregulated in Ikkϵ-deficient tissues, subsequently affecting the recruitment of tumor-associated macrophages and IL17A synthesis. Further studies revealed that IL17A synergized with commensal bacteria to trigger Ikkϵ phosphorylation in transformed intestinal epithelial cells, establishing a positive feedback loop to support tumor development. Therefore, TNF, LPS, and IL17A-dependent signaling pathways converge on Ikkϵ to promote cell survival and to establish an inflammatory tumor microenvironment in the intestine upon constitutive Wnt activation. � 2016 American Association for Cancer Research

The Prosurvival IKK-Related Kinase IKK epsilon Integrates LPS and IL17A Signaling Cascades to Promote Wnt-Dependent Tumor Development in the Intestine

Constitutive Wnt signaling promotes intestinal cell proliferation, but signals from the tumor microenvironment are also required to support cancer development. The role that signaling proteins play to establish a tumor microenvironment has not been extensively studied. Therefore, we assessed the role of the proinflammatory Ikk-related kinase Ikke in Wnt-driven tumor development. We found that Ikke was activated in intestinal tumors forming upon loss of the tumor suppressor Apc. Genetic ablation of Ikke in beta-catenin-driven models of intestinal cancer reduced tumor incidence and consequently extended survival. Mechanistically, we attributed the tumor-promoting effects of Ikk epsilon to limited TNF-dependent apoptosis in transformed intestinal epithelial cells. In addition, Ikk epsilon was also required for lipo-polysaccharide (LPS) and IL17A-induced activation of Akt, Mek1/2, Erk1/2, and Msk1. Accordingly, genes encoding proinflammatory cytokines, chemokines, and anti-microbial peptides were downregulated in Ikk epsilon-deficient tissues, subsequently affecting the recruitment of tumor-associated macrophages and IL17A synthesis. Further studies revealed that IL17A synergized with commensal bacteria to trigger Ikk epsilon phosphorylation in transformed intestinal epithelial cells, establishing a positive feedback loop to support tumor development. Therefore, TNF, LPS, and IL17A-dependent signaling pathways converge on Ikk epsilon to promote cell survival and to establish an inflammatory tumor microenvironment in the intestine upon constitutive Wnt activation. (C) 2016 AACR

Thinking out of the box - New approaches to controlling GVHD

Graft-versus-host disease (GVHD) remains a major limitation of allogeneic hematopoietic cell transplantation (allo-HCT). Despite major advances in the understanding of GVHD pathogenesis, standard GVHD prophylaxis regimens continue to bebased on the combination of a calcineurin inhibitor with an antimetabolite, while first line treatmentsstill relies on high-dose corticosteroids. Further, no second line treatment has emerged thus far in acute or chronic GVHD patients who failed on corticosteroids. After briefly reviewing current standards of GVHD prevention and treatment, this article will discuss recent approaches that might change GVHD prophylaxis / treatment in the next decades, with a special focus on recently developed immunoregulatory strategies based on infusion of mesenchymal stromal or regulatory T-cells, or on injection of lowdose interleukin-2