Volume Abstracts.pmd - Oxygen Club of California

OXYGEN CLUB OF CALIFORNIA UNIVERSITY OF TURIN

2012

OXIDANTS AND ANTIOXIDANTS IN BIOLOGY Cell Signaling and Nutrient-Gene Interactions

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ABSTRACTS

20-23 JUNE 2012 ALBA, ITALY

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ORGANIZERS Giuseppe Poli DEPARTMENT OF CLINICAL AND BIOLOGICAL SCIENCES, UNIVERSITY OF TURIN SAN LUIGI GONZAGA HOSPITAL, ORBASSANO, TURIN, ITALY

Maret G. Traber LINUS PAULING INSTITUTE, OREGON STATE UNIVERSITY, CORVALLIS, OREGON, USA

SCIENTIFIC ADVISORY COMMITTEE Enrique Cadenas DEPARTMENT OF PHARMACOLOGY AND PHARMACEUTICAL SCIENCES, SCHOOL OF PHARMACY, UNIVERSITY OF SOUTHERN CALIFORNIA, LOS ANGELES, CALIFORNIA, USA

César G. Fraga PHYSICAL CHEMISTRY–INSTITUTE FOR BIOCHEMISTRY AND MOLECULAR MEDICINE, SCHOOL OF PHARMACY AND BIOCHEMISTRY, UNIVERSITY OF BUENOS AIRES-CONICET, BUENOS AIRES, ARGENTINA

Klaus Kraemer SIGHT AND LIFE, DSM NUTRITIONAL PRODUCTS LTD., BASEL, SWITZERLAND

Lester Packer DEPARTMENT OF PHARMACOLOGY AND PHARMACEUTICAL SCIENCES, SCHOOL OF PHARMACY, UNIVERSITY OF SOUTHERN CALIFORNIA, LOS ANGELES, CALIFORNIA, USA

Helmut Sies INSTITUTE OF BIOCHEMISTRY AND MOLECULAR BIOLOGY I, FACULTY OF MEDICINE, HEINRICH-HEINE-UNIVERSITY DÜSSELDORF, DÜSSELDORF, GERMANY

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CONTENTS KEYNOTE LECTURE ................................................................................. 5 SESSION I NRF2-DRIVEN REGULATION OF ANTIOXIDANT DEFENSES ............................ 7 SESSION II NUTRIENT-GENE INTERACTIONS AND EPIGENETICS ..................................... 15 SESSION III NOVEL ROLES OF MICRONUTRIENTS ........................................................ 21 SESSION IV LIPID OXIDATION AND SIGNALING ............................................................. 31 SESSION V EPIGENETICS, METABOLISM, AND AGING ................................................... 41 POSTERS ................................................................................................ 57 AUTHOR INDEX .................................................................................... 175 SPONSORS ............................................................................................ 183

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KEYNOTE LECTURE

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Metabolic reprogramming, caloric restriction and aging ROZALYN M. ANDERSON1, JAMIE BARGER2, TOMAS PROLLA3, RICKI J. COLMAN4, AND RICHARD WEINDRUCH1 1

Department of Medicine / VA Geriatric Research, Education and Clinical Center; University of Wisconsin-Madison, WI 53705; 2LifeGen Technologies, LLC, Madison, WI 53719; 3Department of Genetics and Medical Genetics, University of Wisconsin-Madison, WI 53706; 4Wisconsin National Primate Research Center, University of Wisconsin-Madison, Wisconsin 53715

The number of older persons in many nations is rapidly growing and should continue to do so. This demographic reality supports the urgent need for interventions that can extend the span of good health. Caloric restriction (CR) is one such candidate intervention because health span extension has been demonstrated in rodents and rhesus monkeys. Importantly, because it is unlikely that millions of people could adhere to long-term CR, there is great interest in identifying nutrients or drugs that can mimic the effects of CR (so-called “CR mimetics”) in animals/people consuming normal calorie intakes. A mechanistic understanding of CR will facilitate the discovery of CR mimetics. An inverse linear relationship between calorie intake and lifespan in mice suggests that regulators of energy metabolism are important in CR’s actions. Many studies reveal tissue-specific changes in energy metabolism with CR and suggest that metabolic reprogramming plays a critical role in its mechanism of aging retardation. I will discuss data germane to these points derived from my 37 years of studying CR, in collaboration with many gifted colleagues, in mice and monkeys. Lastly, unpublished data based on a transcriptomic meta-analysis revealing conserved pathways to longevity will be presented. The rapid growth of knowledge on the retardation of aging and diseases by CR provides optimism in the search for needed interventions.

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NRF2-DRIVEN REGULATION

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SESSION I DEFENSES

Molecular basis of Keap1-Nrf2 system function MASAYUKI YAMAMOTO1, KEIKO TAGUCHI1, TAKAFUMI SUZUKI1, AND HOZUMI MOTOHASHI1,2 1

Department of Medical Biochemistry and 2Center for Radioisotope Sciences, Tohoku University Graduate School/ School of Medicine, Sendai 980-8575, Japan

Toxic chemicals (often electrophiles) and reactive oxygen species (ROS) activate expression of detoxifying and antioxidant genes through antioxidant/electrophile responsive element (ARE/EpRE). Transcription factor Nrf2 is essential for the coordinated induction of cellular defense enzymes through ARE. This notion is best demonstrated in animal models, showing that Nrf2-null mice are sensitive to a wide variety of electrophiles and ROS. Keap1 is a component of ubiquitin-E3 ligase and degrades Nrf2 constitutively. Electrophiles and ROS liberate Nrf2 from the repression by Keap1 and provoke nuclear accumulation of Nrf2. Keap1 possesses reactive cysteine residues that act as sensors for xenobiotic and oxidative stresses. We refer this system to as the Cysteine Code. Mouse and zebrafish models demonstrate that multiple sensor functions reside within Keap1. The Hinge and Latch model proposed for the Keap1-Nrf2 system describes the mechanism of nuclear accumulation of Nrf2 in a Cul3Keap1 E3 ubiquitin ligase-dependent manner. We have verified this model through structure biology, mouse/zebrafish genetics, and human cancer analyses. In human cancers missense mutations have been identified in KEAP1 and NRF2 genes. These mutations disrupt the KEAP1-NRF2 complex and result in constitutive activation of NRF2. Elevated expression of NRF2 target genes confers advantages on cancer cells. Transgenic mouse models provide evidence that mutated form of Keap1 analogous to cancer genotypes lose the ability to repress Nrf2 in vivo. Thus, the Keap1-Nrf2 system opens a new avenue to the understanding of the signal transduction and regulatory processes underlying the stress response and cancer progression.

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Keap1-Nrf2 signaling: targets for disease prevention THOMAS W. KENSLER 1

1,2

Department of Pharmacology & Chemical Biology, University of Pittsburgh, Pittsburgh, PA 15261 and Department of Environmental Health Sciences, 2 Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA

Health reflects the ability of an organism to adapt to stress. Stressesmetabolic, proteotoxic, mitotic, oxidative and DNA-damage stresses not only contribute to the etiology of cancer and other chronic degenerative diseases but are also hallmarks of the cancer phenotype. Activation of the Kelch-like ECH-associated protein 1 (KEAP1)– NF-E2-related factor 2 (NRF2)-signaling pathway is an adaptive response to environmental and endogenous stresses and serves to render animals resistant to chemical carcinogenesis, other forms of toxicity, and inflammation whilst disruption of the pathway typically exacerbates these outcomes. Protection against these stresses is manifest in multiple ways: (i) prevention of macromolecular damage through induction of electrophile detoxication and antioxidative enzymes, as well as dampening of inflammatory processes, (ii) induction of macromolecular damage repair/removal systems including the proteasome, DNA repair and autophagy, and (iii) activation of tissue repair/regeneration pathways. These cytoprotective effects of Nrf2 reflect responses mediated by direct activation of downstream effector genes and through cross-talk with other signaling networks contributing to cellular plasticity including aryl hydrocarbon receptor, NF-kB, p53 and Notch1. In addition to endogenous signaling molecules such as reactive oxygen and nitrogen species, growth factors and oxidized lipids, the Keap1-Nrf2 pathway can also be induced by thiol-reactive small molecules including dithiolethiones (e.g., oltipraz), isothiocyanates (e.g., sulforaphane) and triterpenoids (e.g., CDDOIm) that demonstrate protective efficacy in preclinical chemoprevention models and in clinical trials. Thus targeting the pathway may provide important opportunities for disease prevention. Supported by NIH grants CA039416, CA094076 and ES006052.

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Nrf2-mediated redox signaling in endothelial cells: consequences for cardiovascular disease GIOVANNI E. MANN Cardiovascular Division, British Heart Foundation Centre of Research Excellence, School of Medicine, King’s College London, 150 Stamford Street, London, SE1 9NH, UK

Fetal exposure to pre-eclampsia (PE) and gestational diabetes (GDM) in utero is strongly associated with a higher risk of cardiovascular disease and insulin resistance in later life, and accumulating evidence suggests this may be a consequence of fetal programming.1 Notably, offspring of mothers with PE or GDM exhibit elevated blood pressure and reduced endothelium-dependent reactivity. We previously reported abnormal NO production, insulin resistance and reduced cell proliferation in fetal umbilical vein endothelial cells (HUVEC) from GDM pregnancies,2,3 and in studies with fetal cells from PE pregnancies identified abnormalities in NO production and regulation of [Ca2+]i.4,5 These phenotypic changes are maintained during culture, highlighting the involvement of fetal programming and epigenetic influences. As in PE and GDM, placental oxidative stress is propagated to the maternal and fetal vasculature via circulating lipid peroxides and H2O2, we hypothesised that sustained oxidative stress in the fetal vasculature impairs endogenous antioxidant defences. We have recently established that activation of the redox sensitive transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2)6 and its downstream target antioxidant enzymes is markedly inhibited in HUVEC from PE or GDM pregnancies. Both cell types exhibited marked deficits in glutathione synthesis, increased basal mitochondrial superoxide production, reduced nuclear translocation of Nrf2, and diminished adaptive increases in the expression of the Nrf2 target genes heme oxygenase-1 (HO-1) and/or NAD(P)H quinone oxidoreductase 1 (NQO1) in response to the lipid peroxidation product 4-hydroxynonenal. A proteomic analysis of normal and GDM HUVEC confirmed the altered GDM phenotype, characterised by markers of increased oxidative stress, reduced antioxidant protection and reduced cell proliferation. The altered phenotype of fetal endothe-

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lial cells derived from PE and GDM pregnancies may well contribute to the increased risk of developing type 2 diabetes and cardiovascular disease in childhood and adulthood.7-9 Supported by BHF 1. Simmons, R. (2005). Trends Endocrinol Metab 16, 390-394. 2. Sobrevia, L., Cesare, P., Yudilevich, D.L., & Mann, G.E. (1995). J Physiol 489, 183-192. 3. Sobrevia, L., Yudilevich, D.L., & Mann, G.E. (1998). J Physiol 506, 219-230. 4. Ishii, T., Itoh, K., E. Ruiz, Leake, D.S., Unoki, H., Yamamoto, M. & Mann, G.E. (2004). Circ Res 94, 609-616. 5. Steinert, J.R., Wyatt, A.W., Poston, L., Jacob, R. & Mann, G.E. (2002). FASEB J 16, 721-723. 6. Steinert, J.R., Poston, L., Mann, G.E. & Jacob, R. (2003). FASEB J 17, 307309. 7. Chapple, S. et al. (2010). Free Radical Res 44, 1125-1171. 8. Gao, L. & Mann, G.E. (2009). Cardiovasc Res 82, 9-20. 9. Cheng, X., Siow, R.C.M. & Mann, G.E. (2010). Antiox Redox Signaling 14, 469-487.

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Adaptive survival response mediated by the Nrf2-induced HO-1 upregulation YOUNG-JOON SURH Tumor Microenvironment Global Core Research Center and WCU Department of Molecular Medicine and Biopharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, South Korea

Living organisms are constantly subjected to diverse types of stress both external and internal sources. While excessive stress leads to necrotic or apoptotic death, moderate amounts of noxious stimuli may render the cells adaptive or tolerant to ongoing or subsequent insults. Such adaptive survival response normally accompanies de novo synthesis of proteins through activation of distinct stress-responsive signaling. The induction of heme oxygenase-1 (HO-1) gene expression represents the first line of cellular defence against oxidative/ nitrosative stress and other noxious stimuli. Experimental models of various diseases including acute inflammation, atherosclerosis, neurodegenerative disorders and cancer have demonstrated that the induction of HO-1 can prevent or mitigate the symptoms associated with these ailments. Nuclear factor E2-related factor-2 (Nrf2) has been identified as a major transcription factor responsible for regulating HO-1 gene expression. Our previous studies have demonstrated that hydrogen peroxide and peroxynitrite induce HO-1 expression through activation of Nrf2 signaling, which confers the cellular cytoprotection. Such adaptive response to oxidative or nitrosative stress was mediated, in part, by 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2). This cyclopentenone prostaglandin activated Nrf2 signaling and subsequently induced expression of HO-1 and other cytoprotective proteins. HO-1 induction is also associated with cytoprotection against inflammatory tissue injuries. Thus, HO-1 induction occurred before inflammatory damage manifests in colonic mucosa following administration of dextran sulfate sodium (DSS) to ICR mice. Some chemopreventive/chemoprotective natural products can induce ARE/ EpRE-driven upregulation of HO-1 expression, thereby fortifying cellular defence against oxidative, nitrosative and inflammatory insults. Supported by the Global Core Research Center (GCRC) grant, National Research Foundation-MEST, Republic of Korea

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Nrf2 target genes are induced under marginal selenium-deficiency REGINA BRIGELIUS-FLOHÉ, MIKE MÜLLER

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ANNA P. KIPP

Department Biochemistry of Micronutrients, German Institute of Human Nutrition Potsdam-Rehbruecke, D-14558 Nuthetal, Germany

Mice were fed a selenium-adequate (0.15 mg Se/kg) or a moderate selenium-deficient (0.086 mg/kg) diet to test consequences for gene expression under a suboptimal selenium supply as it appears to prevail in Europe. Microarray analyses in the colon of these mice identified GPx1 and the selenoproteins W, H, and M as highly sensitive to changes in the selenium state. Most sensitive pathways were protein biosynthesis, and those of inflammation, Delta-Notch and Wnt signaling. Activation of the Wnt pathway was deduced from an increase in the expression of β-catenin, Dvl2, Lef1 and c-Myc accompanied by a decrease in GSK3β. Nrf2 target genes were manually selected and their up-regulation under the selenium-poor diet confirmed by qPCR in the colon and duodenum. Up-regulation of phase II enzymes (Nqo1, Gsts, Sult1b1 and Ugt1a6) was most significant in the colon whereas antioxidant enzymes (Hmox1, Prdx1, Srxn1, and Gclc) increased in both intestinal areas. This might be taken as compensation for the loss of selenoproteins. mRNA of the Nrf2-regulated selenoproteins TrxR1 and GPx2 was higher in the selenium-poor duodenum which would enable their immediate translation upon selenium refeeding. In contrast mRNA of SelW decreased further supporting its sensitivity to limited selenium supply. Possible mechanisms for the obvious activation of Nrf2 under moderate selenium deficiency are discussed in the context of a decrease in GSK3β and TrxR1 under the selenium-poor diet which in an adequate status can turn off the Nrf2 signal.

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SESSION II NUTRIENT-GENE INTERACTIONS AND EPIGENETICS

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Micronutrient genomics - an overview BEN

VAN

OMMEN

TNO, Utrechtseweg 48, Zeist, the Netherlands

We ever more understand the molecular and mechanistic intricacies of micronutrient biological functions and the physiological implications of suboptimal functioning. This has three major implications for establishing trace element requirements: 1) by quantifying the biological function, we automatically include all variability related to micronutrient ADME, mode of action, and all genetic/environmental variation on these processes; 2) if multiple micronutrient influence the biological function (and this is often the case), we can assess the requirements each of these by studying the biological process and its biomarkers; 3) if a micronutrient is affecting multiple biological processes, we can define requirements for each of these processes by quantifying these. A number of initiatives are working along these lines and will be illustrated. Based on these assumptions, the combination of modern analytical technologies, genomics knowledge and network biology can be used to derive personalized requirements of multiple micronutrient. More importantly, this approach may be needed to tackle the real issues in micronutrient: strengthening robustness of challenged populations.

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Functional genetic polymorphisms in genes of choline metabolism: an unexpected link to energy metabolism STEVEN H. ZEISEL University of North Carolina at Chapel Hill, Nutrition Research Institute

It is not widely appreciated that there is significant crosstalk between choline/1-carbon metabolism and energy metabolism. There are several common SNPs in genes of choline and folate metabolism that cause metabolic inefficiencies that affect crosstalk between choline metabolism and energy homeostasis. A number of observations in mice with deleted genes of choline metabolism suggest that such crosstalk occurs. Bhmt-/- mice gained less body weight had reduced adiposity, increased fatty acid oxidation and increased FGF21 signaling. Pemt-/- mice fed a high fat diet had increased energy expenditure and did not gain weight and remained insulin sensitive; these differences disappeared when Pemt-/- mice were supplemented with choline. BMI is significantly lower in women with 2 copies of the minor allele of the PEMT SNP rs12325817 when compared to the women without the SNP. Chdh-/- mice have abnormal mitochondrial structure and function. Mice fed a methionine and choline deficient diet were hypermetabolic, lost weight, and had better insulin sensitivity and glucose tolerance. Betaine administration enhanced insulin sensitivity in diet-induced-obese mice and increased insulin signaling pathways in isolated adipocytes. Both BHMT and choline dehydrogenase (CHDH) activities are decreased by insulin and increased by diabetes in rats. Thus, many different lines of investigation suggest that there is a physiological link between of choline/1carbon metabolism and energy metabolism, insulin sensitivity and body fatness. This work was supported by grants from the NIH (DK55865 and DK36530) 1 To whom correspondence should be addressed: 500 Laureate Way, Kannapolis, NC 28081.Phone: 704-250-5003;Fax: 704-250-5001; E-mail: [email protected].

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The redox basis of epigenetic control FREDERICK E. DOMANN Free Radical & Radiation Research Program, Department of Radiation Oncology, Carver College of Medicine, and Holden Comprehensive Cancer Center,University of Iowa, Iowa City, IA 52242

Epigenetic modifications represent mechanisms by which cells may effectively translate multiple signaling inputs into phenotypic outputs. Recent research is revealing that redox metabolism is an increasingly important determinant of epigenetic control that may have significant ramifications in human health and disease. Numerous characterized epigenetic marks, including histone methylation, acetylation, and ADP-ribosylation, as well as DNA methylation, have direct linkages to central metabolism through critical redox intermediates such as NAD+, S-adenosyl methionine, 2-oxoglutarate, ascorbate, FeII, and molecular oxygen. Fluctuations in these intermediates caused by both normal and pathologic stimuli may thus have direct effects on epigenetic signaling that lead to measurable changes in gene expression through alterations to DNA methylation and/or histone modifications. This lecture will present a survey of the metabolism-sensitive epigenetic enzymes and the metabolic redox processes that participate in their regulation. In addition, we will provide a series of clinically relevant illustrations of the communication between metabolism and epigenetics in the pathogenesis of various human disease states including cancer, diabetes, and neurodegenerative disease. We anticipate that the regulatory mechanisms described here will play increasingly large roles in our understanding of human health and disease as metabolic epigenetics research progresses. Supported by NIH R01 CA115438

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MTHFR genotype and riboflavin: a novel gene-nutrient interaction affecting blood pressure HELENE MCNULTY, J.J. STRAIN, AND MARY WARD The Northern Ireland Centre for Food and Health (NICHE), School of Biomedical Sciences, University of Ulster, BT52 1SA Northern Ireland

Hypertension is estimated to carry an almost 3-fold increased risk of developing cardiovascular disease (CVD), while treating hypertension significantly reduces cardiovascular events, and stroke in particular. One genetic variant under recent investigation in relation to hypertension is the 677C’!T polymorphism in the gene encoding the folate-metabolizing enzyme methylenetetra-hydrofolate reductase (MTHFR). The homozygous mutant (TT) genotype has a reported frequency of 10% worldwide, but can be as high as 32% in some populations. We have been studying the modulating role of the B-vitamin, riboflavin, the cofactor (as FAD) for MTHFR. The variant enzyme is known from molecular studies to become inactive as a result of having an increased propensity to dissociate from FAD, but our earlier work suggested that supplementation with low-dose riboflavin could stabilise its activity in vivo. More recently we found that CVD patients with the MTHFR 677TT genotype (compared to CC or CT genotypes) had significantly higher blood pressure, and more importantly, that blood pressure was highly responsive to riboflavin intervention specifically in patients with the TT genotype. Further investigation of these patients 4 years later showed that those with the TT genotype had remained hypertensive despite marked changes in the number and type of antihypertensive medications being prescribed; with riboflavin administration however there was once again a marked blood pressure-lowering response. Thus riboflavin may offer a targeted strategy for managing hypertension, and potentially modulating the risk of stroke, specifically in this genetically at-risk group. Further work is required to elucidate the mechanism through which this gene-nutrient interaction affects blood pressure.

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Dietary histone deacetylase inhibitors for cancer prevention EMILY HO School of Biological & Population Health Sciences and Linus Pauling Institute, Oregon State University, Corvallis, OR, 97331 USA

The reversible acetylation of histones is an important mechanism of gene regulation. During cancer progression, specific modifications in acetylation patterns on histones are apparent. Targeting the epigenome, including the use of histone deacetylase (HDAC) inhibitors, is a novel strategy for cancer chemoprevention. Recently, drugs classified as HDAC inhibitors, have shown promise in cancer clinical trials. Based on the similarity of sulforaphane (SFN) metabolites and other phytochemicals to known pharmacological HDAC inhibitors, we previously demonstrated that sulforaphane acts as an HDAC inhibitor in the prostate, causing enhanced histone acetylation, derepression of P21 and Bax, and induction of cell cycle arrest/apoptosis, leading to cancer prevention. SFN also causes the down-regulation of both Class I and II HDAC protein expression. These epigenetic effects are specific to cancer cells, not normal cells. The ability of SFN to target aberrant acetylation patterns, in addition to effects on phase 2 enzymes, may make it an effective chemoprevention agent. These studies are significant because of the potential to qualify or change recommendations for high-risk prostate cancer patients and thereby increase their survival through simple dietary choices incorporating easily accessible foods into their diets. These mechanistic pre-clinical studies have provided a strong scientific foundation for on-going human clinical trials.

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NOVEL ROLES

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SESSION III MICRONUTRIENTS

Biomedical action of pomegranate ellagitannins DAVID HEBER, ROBERTO VICINANZA, YANJUN ZHANG,

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ZHAOPING LI

UCLA Center for Human Nutrition, David Geffen School of Medicine at University of California, Los Angeles CA 90095

Pomegranates have been used medicinally for centuries and are popularly consumed as juice. Pomegranate juice (PJ) and extract (PE) contain high molecular weight (ca. 1000 Dalton) hydrolyzable tannins called ellagitanins (ET). The ET are hydrolyzed in the gastrointestinal tract and ellagic acid (EA) appears in the circulation between 30 minutes and 5 hours after consumption of PJ or pomegranate extract (PE). Through the action of human colonic microflora, EA is converted into metabolites including urolithin A (UA), which are then absorbed, transported in the blood, conjugated in the liver, and excreted in glucuronidated form in the urine between 12 and 56 hours after PJ consumption. Consumption of PJ was associated with a significant increase (15 to 54 months) in the PSA doubling time in men with rising PSA after prostatectomy suggesting a direct action on prostate cancer cells. The anti-tumor effects of PE were shown to be due to inhibition of NF-kappaB activation in tumor xenografts. Recent investigations have demonstrated in vitro synergy of EA and UA in human prostate cancer cells and defined unique molecular mechanisms of action. In LAPC-4 human prostate cancer xenografts in SCID mice, there was also evidence of enhanced inhibition of tumor growth. These observations suggest the potential of pomegranate juice and extracts as food ingredients and as dietary supplements in conditions where inflammation and oxidant stress play an important role. Research supported by unrestricted gifts and research grants from POM Wonderful, Inc. Los Angeles, CA

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Zinc and redox signaling in the developing brain SUANGSUDA SUPASAI, JONATHAN NUTTALL,

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PATRICIA I. OTEIZA

Department of Nutrition and Department of Environmental Toxicology, University of California Davis, Davis CA 95616

The developing brain is highly susceptible to fluctuations in zinc availability. Deficits of zinc activate the NMDA receptor, leading to calcium influx, increased oxidant production, alterations in thiol redox homeostasis, and to a redox-modulation of cell signals. Zinc, a redox inactive metal, has been long viewed as a component of the antioxidant network, and growing evidence points to its involvement in redox-regulated signaling. Zinc can contribute to maintain the cell redox balance through its capacity to: i) regulate oxidant production and metal-induced oxidative damage; ii) dynamically associate with sulfur in cysteines, and be released from these associations by reactive nitrogen and oxygen species; iii) regulate glutathione (GSH) metabolism and the overall protein thiol redox status; and iv) to modulate redox signaling either directly or indirectly. In neuronal cells and embryonic brain, zinc deficiency impairs Nrf2 activation and GSH synthesis, and leads to a deregulation of MAPK, NF-κB and STAT signaling pathways. Given the role of Nrf2 in the upregulation of antioxidant defenses, zinc deficits increase the susceptibility of neurons to oxidant stressors. MAPK, NF-κB and STAT play key roles in the decisions of progenitor cells to proliferate, survive, and differentiate into neurons or glial cells. Thus, a suboptimal zinc availability to the fetus, either of nutritional or secondary (maternal exposure to toxicants, infections, chronic diseases) can result in altered brain development.

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Vitamin E in the prevention of cardiovascular disease - the importance of proper patient selection ANDREW P. LEVY Department of Anatomy and Cell Biology, Technion Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel 31096

Vitamin E is a naturally occurring fat-soluble antioxidant which has been proposed as a treatment for both primary and secondary protection against cardiovascular (CV) events. Promising data from observational epidemiological studies associating higher vitamin E dietary intake with lower risk of CV events have not been validated in randomized, controlled clinical trials assessing the effect of vitamin E on CV outcomes. While the pendulum of medical opinion has swung to suggest that high dose vitamin E supplements have no place in the treatment and prevention of CVD, new data is emerging that allows identification of a specific target population for this treatment, namely patients with Diabetes Mellitus and the haptoglobin genotype 2-2. This review details the scientific basis and clinical evidence related to the effect of vitamin E on CV outcomes, and the importance of proper patient selection in gaining therapeutic benefit from this intervention.

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CoQ, adipocytes and metabolism LOUIS CASTEILLA STROMALab, UMR 5273, UPS/CNRS/EFS, Inserm U1031 Toulouse, France

Coenzyme Q (CoQ) is an obligatory partner of mitochondrial biology and the unique lipophilic antioxidant synthesized in humans. Although increasing data underline the importance of mitochondria and oxidative stress in adipocyte biology, few link between CoQ and metabolic disorders has been yet established. We demonstrated that CoQ synthesis is a feature of adipocyte differentiation. Its content is specifically and significantly decreased in adipose tissue of obese mice and humans. Using in vitro model, we showed that the inhibition of CoQ synthesis in preadipocytes impairs adipocyte differentiation, lipid storage, and hypertrophy. The full restoration of its content in adipose tissue of obese mice can be achieved via the coadministration with PPARg ligand. This improves body weight, oxidative metabolism, metabolic parameters and fat functions. Furthermore, this counteracts adverse side effects classically induced by PPARg ligands alone. Finally, we determined a threshold for CoQ content in adipose tissue below which individuals are obese. In another tissue such as liver, High fat diet induces a decrease in CoQ content that is associated with the inhibition of fatty acid oxidation and of mitochondrial oxidative-phosphorylation associated increased mitochondrial ROS production. As a whole, these data demonstrate that CoQ behaves as a key and new regulator of lipid metabolism and that the manipulation of its content could open new therapeutic perspectives.

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Downstream effects of GSH depletion as inducers of longevityassociated pathways MARIA ROSA CIRIOLO Department of Biology, University of Rome “Tor Vergata” and IRCCS San Raffaele, Rome, Italy

GSH is the main non-enzymatic antioxidant implicated in maintenance of redox homeostasis, detoxification and direct scavenging of oxyradicals. GSH has also a pivotal function in buffering the intracellular production of nitric oxide (NO), particularly in brain wherein NO acts as an important neuromodulator and neurotransmitter. Indeed chemical GSH depletion triggers NO unbalance and nitroxidative stress, which is the primary cause of death in proliferating neuroblastoma or embryonic neurons. The importance of GSH in limiting the detrimental effect of NO is corroborated by the evidence that in adult brain even atrivial GSH decrease is able to cause protein nitration. Interestingly, neurodegenerative manifestations are not observed in vivo owing to the connected induction of an adaptive antioxidant response by way of a novel NO/PGC-1alpha-dependent mechanism. Nutrient limitation, a dietary regimen that is generally linked to longevity, induces a prominent efflux of intracellular GSH and redox alterationthat is mandatory for activating NO-driven autophagy. On the basis of these evidence it can be assumed that the decline of GSH level occurring with normal ageing is a beneficial rather than a detrimental event and may serve as a molecular signal inducing a NO-mediated endogenous adaptive response (i.e. antioxidants expression and/or autophagy) that culminates in increased resistance to environmental/pathological stress and possibly in the extension of lifespan.

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Redox modulation of cell signaling by lipoic acid and derivatives DAVID A. CARLSON1, DAO YAO HE2, EKATERINA KALASHNIKOVA1, RINA GENDELMAN1, 3, SARAH J. FISCHER1, AND LESTER PACKER4 1

GeroNova Research Inc, 4677 Meade Street Richmond Ca 94804, 2 C&M Biolabs, 5221 Central Avenue, Suite 8A, Richmond, CA 94804, 3 UCSF Cancer Center, 2340 Sutter Street, N361 San Francisco, CA 94115, 4 Pharmacy and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90089

Lipoic acid (LA) is a chiral, medium chain (C8) fatty acid existing as two enantiomers (RLA and SLA) and a racemic mixture (RS-LA). The only form synthesized in biological systems is RLA. Unsolved problems in LA research include the contributions of stereochemistry and carboxyl substitution toward the mechanisms of action of exogenously applied LA. The enantiomers and racemates of a neutral and a positively charged amide derivative; Lipoamide (LAM) and LA Plus (LAP) were synthesized and compared directly with those of LA. Previous studies with the racemates; RS-LAM and RSLAP and the enantiomer, RLAP indicated similarities and differences relative to LA in vitro. SLAM and SLAP have not been previously evaluated. Widely characterized as “antioxidant” LA and derivatives may function simultaneously and differentially as oxidative, reductive and redox modulators of various signal transduction pathways. Additionally, at sufficient concentrations LA and derivatives may induce stress responses leading to increased adaptation and cellular stress resistance. The effects of R, S and racemic forms of 9 test compounds were studied in multiple signaling pathways in HepG2 cells using a dual-luciferase reporter array. The reporter is a mixture of an inducible transcription factor responsive construct encoding firefly luciferase and an internal control plasmid constitutively expressing Renilla luciferase construct. Each inducible construct monitors changes in the activity of a select transcription factor serving as a marker for activation or suppression of a specific signaling pathway. Preliminary results will be presented of novel, stereoselective and non-stereoselective redox protein targets and pathways differentially affected by the test compounds.

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Vascular oxidative stress and inflammation in atherosclerosis and aging: ameliorating effects of alpha-lipoic acid supplementation BALZ FREI, WEIJIAN ZHANG, LIXIN LI, ANTHONY SMITH, TORY HAGEN

AND

Linus Pauling Institute, Oregon State University, Corvallis, OR 97331, USA

Increased oxidative stress and inflammation have been proposed as etiologic factors for atherosclerotic vascular diseases, for which advanced age is a major risk factor. We have shown that á-lipoic acid (LA) exerts anti-inflammatory effects by inhibiting TNFá or lipopolysaccharide (LPS)-induced endothelial cell and monocyte activation in vitro and LPS-induced acute inflammatory responses in vivo. We also reported that LA inhibits atherosclerotic lesion development in apolipoprotein E (apoE)-deficient and apoE/LDL receptordeficient mice, two well-established animal models of human atherosclerosis. The anti-atherosclerotic effect of LA was paralleled by reduced body weight gain, lower serum and VLDL triglyceride levels, and reduced aortic macrophage accumulation and gene expression of adhesion molecules and pro-inflammatory cytokines. More recently, we reported that dietary supplementation with LA caused a decrease in aortic NADPH oxidase activity in old rats, accompanied by a significant decrease in mRNA levels of NOX4 and vascular cell adhesion molecule-1 (VCAM-1). Furthermore, LA supplementation reversed the age-dependent decrease in aortic SOD activity and the age-dependent increase in plasma levels of monocyte chemotactic peptide-1 (MCP-1). Most of the observed effects of LA supplementation are mediated by the transcription factors, NFkB and Nrf-2, which regulate expression of inflammatory genes and phase II detoxification and antioxidant enzymes, respectively. Mechanistic aspects of this interaction of LA with gene expression will be further discussed. This work was supported by grant 0760018Z from the American Heart Association and NIH Center of Excellence grant P01 AT002034.

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Redox epigenetic modifications by dietary bioactive compounds in inflammation IRFAN RAHMAN Department of Environmental Medicine, University of Rochester Medical Center, Rochester, NY, USA.

Chromatin modifications and epigenetic regulation are critical for regulation of gene expression. Acetylation of histones facilitates gene expression by allowing the access of transcriptional machinery to DNA. Deacetylation of histones suppresses gene expression by closing the transcriptional machinery on promoters. Oxidative stress causes pro-inflammatory gene transcription, which is associated with changes in chromatin remodeling. Steroids are potent anti-inflammatory agents, but their function is lost during oxidative stress leading to steroid resistance. Cigarette smoke-mediated oxidative stress in cell culture (macrophages and epithelial cells) and mouse models were used to test the hypothesis that dietary bioactive compounds reverse steroid resistance via an effect on histone modifications. Oxidants/ cigarette smoke down-regulated the levels and activities of histone deacetylase 2 (HDAC2) and sirtuin 1 (SIRT1) by oxidative posttranslational modifications, and induce gene expression of pro-inflammatory mediators. Cigarette smoke/oxidant stress caused reduced glucocorticoid sensitivity by reducing deacetylases via Nrf2-dependent mechanism. This was associated with the loss in recruitment of deacetylases onto the promoters of pro-inflammatory genes. Resveratrol (phytoalexin, a flavanoid found in red wine) was found to activate SIRT1 and Nrf2, and protected against oxidants/cigarette smoke-induced pro-inflammatory cytokines release. Similarly, curcumin (diferuloylmethane, an active component of spice turmeric) attenuated the reduction in HDAC2, inhibited NF-kB, and reversed the steroid resistance. Hence, overcoming the steroid resistance by dietary bioactive compounds via molecular epigenetic chromatin mechanisms is an important aspect in treatment of chronic inflammatory diseases where oxidative stress occurs. Current knowledge on epigenetics of steroid resistance on cytokine genes involved in chronic inflammation in light of HDAC2/SIRT1-Nrf2, and ways to overcome steroid resistance by dietary bioactive polyphenols based on chromatin modifications will be presented. 29

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LIPID OXIDATION

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SESSION IV AND SIGNALING

Physiological and unregulated membrane lipid oxidation ETSUO NIKI Health Research Institute, National Institute of Advanced Industrial Science & Technology, Ikeda, Osaka 563-8577, Japan

Lipids such as unsaturated fatty acids, cholesterol, and their esters are susceptible to oxidation and oxidized continuously in vivo by free radical-mediated and radical-free mechanisms. Above all, polyunsaturated fatty acid esters are readily oxidized by free radicalmediated chain mechanisms to produce diverse products. Recent lipidomics technology enables us to analyze lipid oxidation products from biological samples accurately and quantitatively. Lipid oxidation products are potentially cytotoxic and modify proteins and DNA bases. Many studies from our groups and others show that the levels of lipid oxidation products are in general associated with the levels of oxidative stress and progress of diseases, although it has not been demonstrated clearly if the lipid oxidation products play a causal role or serve as reliable biomarker of diseases. On the other hand, some lipid oxidation products are produced in vivo on purpose to exert physiological function. Further, lipid oxidation products such as phosphatidylcholine (PC) hydroperoxide, 4-hydroxy-2-nonenal, hydroxyoctadecadieoic acid, lyso PC, and 7-hydroxycholesterol may induce adaptive response and enhance defense capacity. Such adaptive response has been observed in experimental animals as well as cultured cells. However, it should be noted that, unlike ROS/RNS or enzymatic oxidation products which act as signaling messenger in physiological settings, it may be difficult for free radical-mediated lipid oxidation products to function as physiologically important signaling messenger, since it is difficult to program and regulate the formation of free radicals and control many competing reaction pathways.

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Decision-making by macrophages and dendritic cells using RXR heterodimeric receptors to sense their lipid environment LASZLO NAGY University of Debrecen, Medical and Health Science Center

A key issue in the immune system is to generate specific cell types often with opposing activities. We have explored how intracellular lipid signaling in immune cells via a set of transcription factors, regulates cellular differentiation, subtype specification, immune as well as metabolic homeostasis. Peroxisome proliferator-activated receptor γ (PPARγ) is a lipid-activated transcription factor regulating lipid metabolism and inflammatory response in macrophages and dendritic cells (DCs). These immune cells exposed to distinct inflammatory milieu show cell type specification as a result of altered gene expression. We identified a set of mechanisms how inflammatory molecules modulate PPARγ signaling in distinct subsets of cells. Proinflammatory molecules inhibited, whereas interleukin-4 (IL-4) stimulated PPARγ activity in macrophages and DCs. Furthermore, IL-4 signaling augmented PPARγ activity through an interaction between PPARγ and Signal Transducer and Activators of Transcription 6 (STAT6) on promoters of PPARγ target genes, including FABP4. An in silico analysis predicted activation of PPARγ signaling as well as experimental data suggested the presence of alternative macrophage activation specific endogenous PPARγ ligand producing mechanisms. We found three enzymes, which can potentially generate endogenous PPARγ ligands in an IL-4 dependent manner. These are MAOA, ENPP2 and ALOX15 producing 5-methoxy-indole acetate, lysophosphatidic acid (LPA) and 13-hydroxyoctadienoic acid (13-HODE) as well as 15-hydroxyeicosatetraenoic acid (15-HETE), respectively. More recently we have used genomic approaches to map the localization and the cistromic interactions of nuclear receptors and histone marks as well as inflammatory transcription factors. These interactions, underpinning cell type-specific responses represents a unique way of controlling nuclear receptor signaling by inflammatory molecules in immune cells.

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Lipid oxidation-mediated mechanisms of destabilization of the atherosclerotic plaque GABRIELLA LEONARDUZZI

AND

GIUSEPPE POLI

Department of Clinical and Biological Sciences, University of Turin, Orbassano, Torino, Italy

An imbalance in the matrix metalloproteinases/tissue inhibitors of metalloproteinases (MMPs/TIMPs) contributes to atherosclerotic plaque destabilization and rupture. Among MMPs, MMP-9 has been consistently implicated in the pathophysiology of plaque rupture. Oxidative modifications of low density lipoproteins (LDLs), major carriers of cholesterol in human circulation, provide them with several pro-atherogenic properties which might contribute to plaque destabilization. Among the different molecules present in oxidized LDLs and potentially responsible for its bioactivity, there are cholesterol oxidation products, termed oxysterols. To clarify whether oxysterols might play a role in plaque destabilization, we investigated, in human promonocytic U937 cells, the effect of an oxysterol mixture of composition similar to that in advanced human carotid plaques on expression and synthesis of MMP9 and its endogenous inhibitors TIMP-1 and TIMP-2. A marked increment of MMP-9 gene expression and protein levels, but not of its inhibitors, was observed, as well as of MMP-9 gelatinolytic activity. Using antioxidants or specific inhibitors or siRNAs, we demonstrated that the oxysterol mixture induces MMP-9 expression through: i) overproduction of reactive oxygen species (ROS) likely by NADPH oxidase and mitochondria, ii) up-regulation of mitogen-activated protein kinases signaling pathways via protein kinase C, iii) up-regulation of activator protein-1- and nuclear factor-κB-DNA binding. Moreover, oxysterols induce the expression of various inflammatory molecules, such as IL-1β, IL-8 and TNF-α, presumably through TLR4 involvement. These inflammatory molecules appear to contribute to plaque vulnerability by their subsequent induction of MMP9 expression. These results suggest that oxysterols, accumulating in advanced atherosclerotic lesions, significantly contribute to plaque vulnerability by promoting MMP-9/TIMP-1 and TIMP-2 imbalance.

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Oxysterols in the pathogenesis of inflammatory-based diseases LUIGI IULIANO Vascular Biology and Mass Spectrometry Laboratory, Department of MedicoSurgical Sciences and Biotechnology, Sapienza University of Rome, Latina, Italy

Cholesterol, 3β-hydroxycholest-5-ene, is a principal lipid of mammalian cells and is present in all membrane compartments of cells. The addition of oxygen to the cholesterol molecule – via either enzymatic or non-enzymatic mechanisms – results in the formation of derivatives known commonly as oxysterols. In the absence of enzymatic catalysis, oxysterols may be formed when cholesterol reacts with free radical species (i.e. the superoxide/hydrogen peroxide/ hydroxyl radical system) and by non-radical reactive oxygen species (e.g. singlet oxygen, HOCl or ozone). Oxysterols are known to exert a multitude of biological effects of potential pathophysiological relevance, which are dependent on the position of the additional oxygen function in the cholesterol molecule. These effects are mediated by direct biophysical effects on membranes and/or stereospecific interactions with proteins. Oxysterols can be viewed as part of the cellular machinery that governs the integrity of the cell and function by acting at the level of signalling, and translational- and post-translational-gene expression. Key proteins in the control of metabolism reportedly targeted by oxysterols include liver X receptors (LXRs), which exert a key role in lipid metabolism, sterol regulating element binding proteins (SBREPs); oxysterol-binding proteins (OSBP); estrogen receptor; Nieman Pick disease proteins (NCP1, NCP2); Hedgehog system; and Epstein-Barr virus induced gene 2 (EBI2). The pace of discovery in the field of oxysterols is accelerating rapidly and oxysterols of valuable potential importance, including 3β-hydroxy-5-oxo-5,6-secocholestan-6-al (i.e., 5,6-secosterol) and its carboxyaldehyde generated in close proximity of activated inflammatory cells, are emerging as putative effectors and biomarkers of inflammation-oxidative stress processes.

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Electrophilic nitro-fatty acids as anti-inflammatory mediators in the vascular compartment FRANCISCO J. SCHOPFER Department of Pharmacology & Chemical Biology, University of Pittsburgh, Pittsburgh, PA 15261, USA

Nitration of unsaturated fatty acids by oxides of nitrogen yield electrophilic derivatives that modulate protein function via post-translational protein modifications. The electrophilicity is conferred by the conjugated nitroalkene group, making them highly reactive with thiols and histidines through Michael addition reactions. Nitrated fatty acids are formed endogenously during ischemia and reperfusion, inflammatory conditions and gastric acidification from addition reactions between nitrogen dioxide and conjugated linoleic acid (CLA). Conjugated linoleic acid is reported to be the primary endogenous substrate for fatty acid nitration in vitro and in vivo resulting in up to 105 greater yields of nitration products when compared to bis-allylic linoleic acid. In particular, two main isomers are formed by mitochondria, activated macrophages and gastric acidification that activate PPARgamma, react with KEAP1 to turn on Nrf2 signaling, activate heat shock responses and inhibit Nf-kB. Nitroalkene derivatives of CLA and their metabolites are detected in the plasma and urine of healthy humans and are increased in tissues undergoing episodes of ischemia-reperfusion. Dietary CLA and nitrite supplementation in rodents elevates NO2-CLA levels in plasma, urine and tissues and induces electrophile-regulated HO-1 expression in the colonic epithelium. These results affirm that metabolic and inflammatory reactions yield electrophilic signaling mediators that modulate cellular adaptive responses and lead to tissue protection and resolution of inflammation.

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High cholesterol diet-induced impairment of glucose homeostasis in mice with fatty liver: role of inducible NOS OREN TIROSH Institute of Biochemistry, Food Science and Nutrition, The Robert H Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel

This study was designed to elucidate the role of inducible nitric oxide synthase (iNOS) in septic fatty livers with the accentuation on glucose metabolism. Wild-type (WT) and iNOS-knockout (iNOS-/) mice were fed with high-cholesterol diet (HCD) (1%w/w) for 6 weeks. Following diet period, mice were injected with LPS (5 mg/ Kg) to induce endotoxemia. Chronic consumption of HCD led to a significant increase in hepatic steatosis in WT and iNOS-/- mice. LPS administration caused marked liver damage which was further exacerbated in the absence of iNOS. Enhanced liver injury in iNOS/- mice was in association with a severe hypoglycemia. In the knockout group, glycogen contents were significantly higher while Hypoxia-induciable factor-1 (HIF1) signaling was markedly attenuated compared to control WT. Conversely, LPS treatment equally suppressed the expression of key gluconeogenic enzymes. The role of iNOS and HIF1 in hepatic glucose metabolism was further confirmed in vitro. Hepatic glucose output was augmented by overexpression of HIF1 or by using NO-donor in AML-12 hepatocytes. Results also demonstrated increased oxidative stress and reduced heme oxygenase-1 (HO-1) mRNA in the livers of iNOS-/- mice. Furthermore, the amounts of plasma tumor necrosis factor-α (TNFα) and intrahepatic TNFα mRNA were significantly elevated in the absence of iNOS. Conclusion: these data highlights the essential role of iNOS in the glycemic response to LPS in fatty livers and further argues for the beneficial effects of iNOS in these livers.

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Non-alcoholic fatty liver disease, oxidative stress, and mitochondria GAETANO SERVIDDIO C.U.R.E. Centre for Liver Diseases Research and Treatment, Department of Medical and Occupational Sciences, University of Foggia, Italy

Non-alcoholic steatohepatitis (NASH) is an increasingly recognized condition that may progress to end-stage liver disease. NASH pathogenesis is not well understood: Day and James proposed a double hit model, where steatosis represents the first hit, making the liver vulnerable to a second hit, which leads to hepatic inflammation and fibrosis. Oxidative stress, lipid peroxidation and cytokines are considered the best candidates for the role of second hit. Several recent studies have revised this model of pathogenesis assigning to oxidative stress a central role and limiting steatosis to being epiphenomenon of the injurious mechanism. Oxidative stress in NASH is closely related to mitochondrial dysfunction. We have defined that, during NASH progression, oversupply of free fatty acids induces increase of mitochondrial H2O2 production that in turn oxidizes mitochondrial membranes and regulates activity of the Uncoupling Protein 2 (UCP2) and Carnitine Palmitoyl Transferase 1 (CPT1). Mitochondria play a key role in hepatocytes metabolism, being the site of âoxidation and oxidative phosphorylation. We and others demonstrated that mitochondrial abnormalities are closely related to the pathogenesis of NAFLD, which raises the possibility that NAFLD is a mitochondrial disease. At present, there is no proven therapy for NASH, and pharmacotherapy is an area of active research. The introduction of drugs directly able to reduce oxidative stress, in association with clinical strategies directed at lowering lipid accumulation, would be important in the treatment of these disorders. Recent approaches in mitochondrial drug delivery systems and mitochondria-targeted molecules would be potentially effective in the targeting of mitochondrial dysfunction in NASH.

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Lipid oxidation products: signalling and biomarkers for inflammatory disease in humans ANA REIS1, NORSYAHIDA MOHD FAUZI2, ANDREW R. PITT1 AND CORINNE M. SPICKETT1 1 School of Life and Health Sciences, Aston University, Birmingham, B4 7ET, UK; 2Strathclyde Institute of Pharmacy and Biomedical Science, University of Strathclyde, Glasgow, G4 0NR, UK

Phospholipid oxidation occurs in inflammation and generates a wide range of different products, including intact esterified oxidized phospholipids, chain shortened phospholipids and non-esterified alkenal products, as well as chlorinated and nitrated lipids. Biological effects such as toxicity, cell-cell adhesion, ROS and cytokine production have been reported for several of these products and suggested to play important roles in many diseases involving oxidative stress. We have shown previously that oxidized 1-palmitoyl-2arachidonoyl-sn-glycero-3-phosphocholine oxPAPC, a model of oxLDL) does not induce classical activation of the pattern recognition receptors TLR2 and TLR4 leading to NFkB activation, and indeed can inhibit LPS or bacterial lipoprotein-induced signalling via these receptors. In contrast, chlorinated lipids do not appear to affect LPS-induced inflammatory processes, having little effect on cytokine production in myeloid cells. oxPAPC products such as 1-palmitoyl2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), which contains an aldehyde group, can form adducts with proteins and alter their biological function. Modern mass spectrometry methods are facilitating both lipidomic and proteomic analysis of LDL. We have developed targetted LCMS and LCMS ion scanning methods to facilitate the analysis of oxidized and chlorinated phospholipids and detect glycosylation and alkenal adducts on albumin and ApoB-100. This approach is being applied to study LDL changes in cardiovascular and related inflammatory diseases. A combination of lipidomics and proteomics presents a powerful approach to understanding lipid signalling and identifying biomarkers in inflammatory diseases.

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Protein oxidation: reducing the toxic effects TILMAN GRUNE Department of Nutritional Toxicology, Institute of Nutrition, Friedrich Schiller University Jena, 07743 Jena, Germany

Proteins which are oxidatively modified are degraded by the 20S proteasome in an ATP- and ubiquitin-independent pathway. However, in several studies an enhanced ubiquitination of yet unknown proteins was reported. By determination of oxidized- and ubiquitinated proteins we demonstrated that the oxidized proteins are not preferentially ubiquitinated. Identification of the ubiquitinated proteins revealed a number of metabolic and regulatory proteins to be ubiquitinated together with a number of chaperons. This reveals a close interaction on the function of chaperones and the ubiquitin/ proteasome system. Interestingly, in the case of strong oxidative stress, if the proteasomal system is overwhelmed and inhibited an up-regulation of several heat shock proteins (Hsps), including Hsp27, Hsp70 and Heme Oxygenase-1 (HO-1) takes place. It was demonstrated that the induction of classical Hsps, such as Hsp27 and Hsp70, is dependent on an HDAC6-dependent mechanism. Our data demonstrate that also HO-1 induction is mediated by HDAC6 via p38MAPK deacetylation and Nrf-2 activation. If the proteasomal system is overwhelmed and the chaperones are not sufficient to maintain the solubility of oxidized proteins, these proteins aggregate and form lipofuscin. In the current literature, the lysosomal system is considered to be essential in the intracellular formation and accumulation of lipofuscin. In contrast to that, our experimental results demonstrated that both autophagosomes and the lysosomal system are not mandatory for the formation of lipofuscin. However, an inhibition of these systems is not leading to a decline in the lipofuscin formation, but rather in an enhanced toxicity of the formed protein aggregates.

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SESSION V EPIGENETICS, METABOLISM, AND AGING

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Epigenetic regulation of aging MARIO F. FRAGA1,2 1 Department of Immunology and Oncology, National Center for Biotechnology, CNB-CSIC, Cantoblanco, Madrid E-28049, Spain 2 Cancer Epigenetics Laboratory, Instituto Universitario de Oncología del Principado de Asturias (IUOPA), HUCA, Universidad de Oviedo, Oviedo, Spain

Epigenetics of aging is an emerging field that promises exciting revelations in the near future. Epigenetic mechanisms, including DNA methylation and histone modification, are determinants of normal development and can change during aging. Some of the epigenetic alterations described during aging, as hypermethylation at specific promoters and decrease of global DNA methylation, are also associated with tumor development. The epigenetic alterations occurring during development and aging can be stochastic and depend on genetic (intrinsic) and environmental (extrinsic) factors. Future challenges in the field involve the determination of the real role of these epigenetic alterations in the establishment of aging-associated phenotypes.

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Epigenetic oxidized redox shift theory of aging (EORS) in brain GREGORY J. BREWER1,2, M.P. WALKER2, K.R. LEVAULT2 , AND DEBOLINA GHOSH2 1

Departments of Neurology and 2Medical Microbiology, Immunology and Cell Biology, Southern Illinois Univ. Sch. Med., Springfield, Illinois, USA 62794-9626

Our 2010 epigenetic oxidized redox shift theory of aging (EORS) proposed to revise the ROS theory of aging with an epigenetically enforced metabolic redox shift that leads to a bioenergetic downward spiral. Since neuron loss, epigenetic changes, less glutathione (GSH) and increased ROS are associated with aging and Alzheimer’s Disease (AD), we wanted determined the relative importance of each of these to neuron death. In the brains of aging and a AD- model mice, we found reversible epigenetic histone modifications and Bdnf expression (Age 2012). We also found an oxidative redox deficit in aging mice and AD-mouse neurons, as well as lower NADH regenerating capacity and GSH levels, even before ROS-mediated macromolecular damage and elevated ROS levels (J. Neurosci. 2012). Here, our strategy imposed a stress of incremental GSH depletion +/ - incremental neuroprotection. GSH depletion increased neuronal death of AD-mouse neurons at increasing rates across the age-span, while non-transgenic neurons were resistant to GSH depletion until old age. Remarkably, the rate of neuron loss with ROS did not increase in old age and was the same for both genotypes, which suggests that cognitive deficits in the AD-model are not caused by ROS. In the opposite approach of neuroprotection, activation of the redox sensitive Nrf2-ARE along with the NAD+-precursor, nicotinamide increased neuron survival, decreased ROS and increased GSH and NAD(P)H in an additive manner. In summary, the reversible redox environment of GSH and NAD(P)H is more important to neuron survival than ROS in aging. Supported by NIH RO1 AG032431

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Aging, single-cell methylomes SILVIA GRAVINA

AND

JAN VIJG

Department of Genetics Albert Einstein College of Medicine, 1301 Morris Park Ave Bronx, New York 10461

Recent data suggest that the epigenome is highly dynamic and serves as an interface between the environment and the inherited static genome. The large volume of epigenomic events and its continuous need of maintenance, i.e., after DNA repair or replication, suggest a high chance of errors. The question we wish to address is how unstable the epigenome really is. Do epimutations accumulate with age and do they occur in a random fashion, i.e., as ‘epigenomic drift’? Do they ever reach levels that are high enough to have functional consequences? To experimentally determine epigenetic drift we focused on DNA methylation, a major layer of epigenomic control. To study intraorgan variation in DNA methylation during aging, it is necessary to have access to procedures that allow assessing DNA methylation patterns at the level of single-cells or single-molecules. Such methodology is currently entirely lacking: to fill this void we have optimized bisulfite sequencing for single cell analysis. The procedure developed allows us to analyze DNA methylation patterns in single cells, within promoter regions of genes or genomewide. Data on mouse fibroblasts, neuronal nuclei and hepatocytes will be presented and discussed. We are currently applying the method to test the hypothesis that random DNA methylation changes accumulate in the mouse brain during aging, contributing to functional decline of somatic cells that gives rise to chronic pathology and aging.

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Selective autophagy on the cellular energetic balance ANA MARIA CUERVO Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY

Autophagy mediates the digestion of cytosolic material in lysosomes. All types of autophagy fulfill two important functions in mammalian cells, serving both as an alternative source of energy, when nutrients are scarce, and as an efficient mechanism for the removal of any intracellular damaged structures. In this talk, I will focus on the connections between selective autophagy and cellular metabolism. Prolonged starvation has been classically known as the best stimuli for chaperone-mediated autophagy (CMA) but the contribution of this pathway to cellular metabolism has not been studied in depth. Working with transformed cells in culture, we have found that blockage of CMA results in a marked decrease in intracellular ATP content. To our surprise, reduced ATP seems to mainly resulting from alterations in main intracellular metabolic pathways. We have found that both glycolysis and beta-oxidation are compromised upon CMA blockage. I will discuss the molecular components identified until date to participate in this effect of CMA over these metabolic pathways. In addition, I will also comment on the reverse situation, namely the impact that different macromolecules incorporated with the diet have on CMA. On this respect we have identified a regulatory effect of nutritional lipids on CMA and have elucidate the molecular mechanisms behind this effect. The active interplay between nutrients and CMA could be of particular importance on conditions as aging and age-related disorders in which CMA activity has been shown to be compromised.

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Modulation of aging genes: importance in longevity and ageassociated frailty JOSÉ VIÑA, CONSUELO BORRÁS JUAN GAMBINI, KHIRA MOHAMED, JUAN-ANTONIO AVELLANA, LEOCADIO RODRIGUEZ-MAÑAS, AND EVA SERNA Departamento de Fisiología, Facultad de Medicina, Universidad de Valencia; Servicio de Geriatría. Hospital de la Ribera, Alzira y Red Española de Envejecimiento y Fragilidad RETICEF

The concept of longevity associated genes received serious support after a review by Sinclair and Guarente (1). Many genes have been associated with longevity. In our laboratory, we have studied some of these. For instance we could attribute the increased longevity in females to the estrogen- dependent over-expression of MnSOD and Glutathione peroxidase (2). Other genes that we have shown to be involved in longevity are p53 (3); p16/Arf (4) RAS-Grf (5) and telomerase (6). We have recently observed that, in humans, oxidative stress is associated more with frailty than with age itself. An obvious approach is to find physiological, nutritional or pharmacological intervention to promote the activity of longevity associated genes. Exercise is a paradigmatic intervention to promote such genes (7) We have recently observed that centenarians, not only reach extreme longevity but also display remarkable low levels of frailty. Thus we decided to search for longevity genes that could be over expressed in centenarians. We have performed the whole mRNAi-ome and correlated it with the transcritome in centenarians (100 ± 2), octogenarians (80- 85) and young persons of the area of Alzira (Valencia, Spain) The main conclusions are: 1. The Principal Component Analysis (PCA) of the mRNAi-ome of centenarians is remarkably similar to that of young persons and different from octogenarians 2. Centenarians up-regulate the expression of mRNAi when compared with young persons. Octogenarians down regulated it. This

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shows that transcription in centenarians is much more regulated than in octogenarians. 3. Comparison of the variations in miRNA-ome and whole transcriptome of centenarians has allowed us to identity mitochondrial proteins that are specifically up regulated in centenarians. References 1 Sinclair DA, Guarente L. Sci Am. 2006 Mar;294(3):48-51, 54-7 2 Borrás C, et al. Free Radic Biol Med. 2003 Mar 1;34(5):546-52 3 Matheu A, et al. Nature. 2007 Jul 19;448(7151):375-9 4 Tomás-Loba A, et al. Cell. 2008 Nov 14;135(4):609-22 5 Matheu A, et al Aging Cell. 2009 Apr;8(2):152-61 6 Borrás C, et al. Aging (Albany NY). 2011 Mar;3(3):262-76 7 Viña J, Gomez-Cabrera MC, Borras C. Br J Nutr. 2007 S36-40

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Redox regulation of cellular stress response in aging and neurodegenerative disorders: role of hormesis and vitagenes VITTORIO CALABRESE1, CAROLIN CORNELIUS1, ANGELA TROVATO SALINARO1, AND EDWARD J. CALABRESE2 1 Clinical Biochemistry, Faculty of Medicine, University of Catania, Italy, Environmental Health Sciences Division, School of Public Health, University of Massachusetts, Amherst, MA, USA

2

Protein quality control is a critical feature of intracellular homeostasis1. In addition, modulation of endogenous cellular defense mechanisms via the stress response signaling represents an innovative approach to therapeutic intervention in diseases causing chronic tissue damage, such as neurodegeneration and cancer1. Protein thiols play a key role in redox sensing, and regulation of cellular redox state is crucial mediator of multiple metabolic, signalling and transcriptional processes. Under optimal conditions long-term health protection is accomplished by protein homeostasis, a highly complex network of molecular interactions that balances protein biosynthesis, folding, translocation, assembly/disassembly, and clearance2,3. Efficient functioning of maintenance and repair processes is crucial for both survival and physical quality of life. This is accomplished by a complex network of the so-called longevity assurance processes, which are composed of several genes termed vitagenes4-8. The term vitagenes refers to a group of genes which are strictly involved in preserving cellular homeostasis during stressful conditions. The vitagene family is actually composed of the heat shock proteins (Hsp) Hsp32, Hsp70, the thioredoxin system and the sirtuin system9. Dietary antioxidants, such as polyphenols and L-carnitine/acetyl-L-carnitine, have recently been demonstrated in vitro to be neuroprotective through the activation of hormetic pathways, including vitagenes1012 . Over the past decade there has been a remarkable increase of interest in hormesis as a result of more significance being given to low dose effects and the use of more powerful study designs which have enabled to identify rational approaches to detect hormetic biphasic dose responses in the low dose zone. The hormetic dose–response, challenging long-standing beliefs about the nature of the dose–response in a lowdose zone, has the potential to affect significantly the

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design of pre-clinical studies and clinical trials as well as strategies for optimal patient dosing in the treatment of numerous diseases, including oxidant disorders. Given the broad cytoprotective properties of the heat shock response there is now strong interest in discovering and developing pharmacological agents capable of inducing stress responses. We have recently focused our research on the role of acetylcarnitine in the defence mechanisms against cellular stress and neurodegeneration. In addition, with a redox proteomics approach, we identified mitochondrial oxidatively modified proteins as a function of brain aging, specifically in those brain regions, such as cortex and hippocampus, that are commonly affected by the aging process. In all brain regions examined, many of the identified proteins were energy-related, such as pyruvate kinase, ATP synthase, aldolase, creatine kinase, and a-enolase. These alterations were associated with increased expression of Hsps, as well as carnosinase and thioredoxin reductase and with significant changes in both cytosolic and mitochondrial redox status in all brain regions analyzed. This findings are relevant to potential pharmacological interventions in healthy medicine strategy, pointing to maximize cellular stress resistance of the brain thus providing neuroprotection9-12, and will be extended to other systemic oxidant disorders such as diabetes and cancer. 1. Halliwell B. (2008) Arch Bioch Biophys 476:107. 2. Calabrese V. (2007) Nature Neurosci 8,766. 3. Calabrese V. (2011) Mol Aspects of Med 32:279. 4. Calabrese V. (2011) Chem Biol 18:1355. 5. Calabrese V. (2011) Mol Aspects of Med 32:258. 6. Calabrese V. (2012) BBA 1822:75388. 7. Calabrese V. (2010) Antiox Redox Signal 13:1763. 8. Calabrese V. (2010) Neurochem Res 35:1880. 9. Calabrese E.J. (2012) Biogerontology epub 10. Calabrese E.J. (2010) HET 29:980. 11. Calabrese V. (2012) BBA 1822:729. 12. Mattson M.P. (2010) Neuron 67:900.

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MitoSENS: allotopic expression of mitochondrial genes using a co-translational import strategy MATTHEW S. O’CONNOR SENS Foundation Research Center, Mountain View, CA 94041

The mitochondrion contains its own genome and encodes 13 proteins that are essential for electron transport chain function and ATP synthesis to function properly. Congenital mutations in many of the mitochondrial genes are the cause of serious disease phenotypes including diabetes, blindness, dementia, ataxia, epilepsy, and many other neurological disorders. Somatic mutations also accumulate in the mitochondria with normal aging. Allotopic expression of mitochondrial genes in the cell’s nucleus is one approach to rescuing mitochondrial mutations. In our strategy, we utilize 5’ and 3’ elements to target the mRNA to the mitochondria; this approach is a variation of allotopic expression and is hypothesized to result in the co-translational import of these proteins into the mitochondria. This approach is hypothesized to overcome the obstacles experienced by many groups in the import of allotopically expressed proteins into the mitochondria, such as clogging of the mitochondrial import machinery by hydrophobic proteins. Thus far, we have stably transfected 5 of the 13 mitochondrial genes into the nuclear genome of human cell lines and are characterizing the expression and function of these exogenously expressed genes. We will discuss current progress and future plans for replacing and/or making redundant the entire mitochondrial genome. We will also discuss potential applications of the MitoSENS approach in treating mitochondrial diseases, as well as the diseases and pathologies of aging.

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Ageing - from molecules to hormesis SURESH RATTAN Laboratory of Cellular Ageing, Department of Molecular Biology and Genetics, Aarhus University, Denmark

Ageing can be understood at various levels, from evolutionary and biological levels to psychological and sociological levels. At the molecular biological level ageing is characterized by the stochastic occurrence and progressive accumulation of molecular damage. Failure of homeodynamics, increased molecular heterogeneity, altered cellular functioning and reduced stress tolerance are the determinants of health status, probability of diseases and the duration of survival. The inefficiency and imperfection of the maintenance and repair systems underlie the biological basis of ageing. Two major issues yet to be resolved in biogerontology are determining the physiological relevance of various types of molecular damage, and establishing the nature of healthy and unhealthy gene-protein networks. Gene therapy, stem cells, and modulation through functional foods, nutriceuticals, cosmeceuticals and other life style alterations are examples of ageing interventions. A promising healthy-ageing approach is that of hormesis by strengthening the homeodynamic ability of self-maintenance through transient and repetitive mild stress-inducing hormetins. Achieving the goal of extended health-span will depend on elucidating and exploiting successful and dynamic interactions among biological, psycho-social and environmental factors.

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Proteolytic signaling dysfunction in aging KELVIN J.A. DAVIES Ethel Percy Andrus Gerontology Center of the Davis School of Gerontology, and Division of Molecular & Computational Biology, Department of Biological Sciences of the Dornsife College of Letters, Arts & Sciences, University of Southern California, Los Angeles, California 90089-0191, USA

Proteins are major targets for oxidative damage, and both the intracellular and extracellular accumulation of oxidized proteins has been reported in many ageing and disease models. In young and healthy individuals, moderately oxidized soluble cell proteins are selectively and rapidly degraded by the Proteasome in the cell cytoplasm, nucleus, and endoplasmic reticulum. Severely oxidized, aggregated, and cross-linked proteins, however, are poor substrates for degradation and actually inhibit the Proteasome. Some mildly aggregated and cross-linked proteins are degraded by autophagy, but more severely aggregated and cross-linked proteins seem to accumulate as inclusion bodies or lipofuscin inside lysosomes. It is, therefore, vitally important that cells rapidly and selectively degrade mildly oxidized proteins, before they undergo more severe oxidation, aggregation, and cross-linking. Young mammalian cells can readily adapt to mild oxidative stress such that they become (temporarily) much more resistant to oxidative damage. Such adaptive responses include the immediate disassembly of the 26S proteasome (catalyzed by HSP70) to form free 20S proteasome and 19S regulator complexes, at which point and ATP/Ubiquitin-dependent proteolysis is temporarily lost. The additional free 20S Proteasomes are of immediate help in degrading oxidized proteins. Next, over a 3-20 hour period, new 20S Proteasomes, Immunoproteasomes, and both 11S (PA28) and PA200 proteasome regulators are synthesized, partially under the control of the Nrf2 signal transduction pathway. We have now obtained similar findings in C. elegans worms, and D. melanogaster fruit flies. The original 26S Proteasomes are re-assembled, and ATP/Ubiquitin-dependent proteolysis is restored. Recent work suggests that certain proteasome subunits and regulators should be classified as true stress proteins. Induction of Proteasome expression provides significantly greater capacity to remove oxidized proteins, and such adaptive re-

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sponses contribute to overall stress resistance. In older cells, however, Proteasome activities decline, and adaptational responses mediated by Nrf2 become sluggish or even ineffectual. In some cases, the antagonistic effects of Nrf1 may actually blunt adaptive proteolytic responses to stress in older cells and organisms. Studies in animals and humans suggest that declining Proteasome activities and, perhaps, declining responsiveness to stress, may contribute to the ageing process, and to various age-associated diseases.

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Reactive oxygen species in muscle wasting during aging MALCOLM J. JACKSON, GIORGOS SAKELLARIOU, TIM PEARSON, APHRODITE VASILAKI, ANNA KAYANI, SIOBHAN SCULLION, AND ANNE MCARDLE Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool L693GA, UK

Chronic loss of skeletal muscle mass and function is a major contributor to frailty and weakness in the elderly. The major cause of age-related loss of muscle mass is a decrease in the number of skeletal muscle fibres associated with atrophy and weakness of the remaining fibres. A large number of studies have reported a dysregulation of reactive oxygen species (ROS) homeostasis during ageing that may potentially lead to increased oxidative damage to tissues and/or to defective redox signaling. Skeletal muscle from aged mammals contains increased amounts of oxidative damage, but whether this is the cause of age-related deficits in muscle or a consequence of ageing has been the subject of controversy. Studies from our group have examined the changes in ROS generation that occur with ageing in man and experimental models and have also utilised a transgenic approach to modify ROS generation in model organisms. Data from these experiments support the hypothesis that aberrant ROS regulation plays a role in the decline in skeletal muscle and in motor neurons that occur during ageing. The key processes involved do not appear to involve merely oxidative damage to tissues, but are associated with defects in important redox-sensitive adaptive responses to contractile activity. This research is supported by the Medical Research Council, Biotechnology and Biological Sciences Research Council and United States National Institute on Aging.

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Testosterone, estrogens and inflammation: a clue for the understanding of frailty in older people? LEOCADIO RODRIGUEZ-MAÑAS1,2, LAURE CARCAILLON2, CARMEN BLANCO3, JESUS F. TRESGUERRES4, GONZALO GUTIÉRREZ-AVILA5, AND FRANCISCO JOSE GARCIA-GARCIA6 1

Department of Geriatrics, Hospital Universitario de Getafe, Madrid, Spain; 2 Fundación para la Investigación Biomédica, Hospital Universitario de Getafe, Madrid, Spain; 3Department of Clinical Analysis, Hospital Universitario de Getafe, Madrid, Spain; 4Department of Physiology, School of Medicine, Universidad Complutense de Madrid, Spain; 5Department of Epidemiology and Public Health, Health Department of Castilla-La Mancha, Toledo, Spain; 6Department of Geriatrics, Hospital Virgen del Valle, Complejo Hospitalario de Toledo, Toledo, Spain

Age-associated changes in sexual hormones represent one of the potential mechanisms leading to frailty. We studied the association between testosterone, estrogen and frailty and assessed sex-differences in the relationship between hormones and frailty. We used cross-sectional data from the Toledo Study for Healthy Aging, a population based cohort study of Spanish elderly. Frailty was defined according to Fried’s approach. Multivariate odds-ratios (OR) and 95% confidence intervals (CI) associated with total testosterone (TT), free testosterone (FT) and estradiol (E2) levels were estimated using polytomous logistic regression. In women, there was a U-shaped relationship between FT levels and frailty (p for FT²=0.03), confined to obese women (p-value for interaction=0.05). In men, the risk of frailty linearly decreased with testosterone levels: adjusted OR =2.9 (95%CI, 1.6-5.1) and 1.6 (95%CI, 1.0-2.5), for 1 SD decrease in TT and FT, respectively. No interaction was found with BMI. Higher E2 levels were associated with frailty among women younger than 79 years old but not in the oldest group (p interaction=0.047). After adjustment, OR of frailty associated with 1 standard deviation increase of E2 was 1.51 (1.04-2.20), p=0.03. We identified an interaction between E2 and hs-CRP on the prevalence of frailty (p-value=0.042). Women having both higher E2 and hs-CRP had an age-adjusted OR of 4.2 (1.7-10.5), p=0.002, compared with women with low levels of both E2 and hs-CRP. There is a relationship between sexual hormones and frailty both in older men and women. However its relevance and its interactions with other mechanisms differ by gender. 55

Oxidative stress and the link of pathophysiology of Alzheimer’s disease with vascular pathology M. CRISTINA POLIDORI1, LUDGER PIENTKA1 1

AND

PATRIZIA MECOCCI2

Department of Geriatrics, Marienhospital Herne, Ruhr University Bochum, Germany, 2Department of Gerontology and Geriatrics, Perugia University Hospital, Perugia, Italy

Vascular risk factors (VRF) like hypertension, diabetes and atherosclerosis linked to dementias and Alzheimer’s disease (AD) are known to cause ischemia- and inflammation-related oxidative stress as well as subsequent reperfusion-related free radical production. Oxidative stress is known to occur early in the onset of cognitive impairment, but the mutual relationships between oxidative stress, vascular comorbidities and cognitive impairment progression are poorly understood. The longitudinal evaluation of elderly patients with mild cognitive impairment and AD with or without vascular comorbidities (groups MCI plus and AD plus, respectively) shows that AD and control subjects have significantly higher plasma levels of carotenoids, tocopherols and retinol than MCI Plus and AD Plus patients. The results are independent of age, gender, BMI as well as intake of fruits and vegetables. In a subset analysis of MCI, AD, MCI Plus and AD Plus subjects, baseline plasma levels of F2 isoprostanes prove to be in average 20% higher in MCI Plus and AD Plus patients whose neuropsychological scores worsened after two years than F2 isoprostane plasma levels in MCI and AD patients neuropsychologically stable at follow up. Vascular comorbidities and risk factors as well as biomarkers of oxidative damage should be early identified in the elderly and always evaluated in aged patients with cognitive impairment to allow primary and secondary lifestyle-based preventive strategies.

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POSTERS

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Natalizmab caused changes in Nrf2 in patient with multiple sclerosis EDUARDO AGÜERA1, FERNANDO SÁNCHEZ-LÓPEZ1, ANA I. GIRALDO2, ALEYDA PÉREZ-HERRERA3, AND ISAAC TÚNEZ2 1

Servicio de Neurología, Instituto Maimónides de Investigación Biomédica de Córdoba IMIBIC/Hospital Universitario Reina Sofía, Córdoba, Spain; 2 Departamento de Bioquímica y Biología Molecular, Facultad de Medicina / IMIBIC/Universidad de Córdoba, Spain, 3Lipids and Atherosclerosis Research Unit, Reina Sofía University Hospital, University of Cordoba IMIBIC/Reina Sofía University Hospital University of Cordoba, Spain and CIBER Fisiopatología de la Obesidad y Nutrición (CIBEROBN), Instituto de Salud Carlos III, Spain

Multiple sclerosis (MS) is an inflammatory demyelinating disease disease of central nervous system (CNS). A recent study of our group has shown that MS presents a peripheral oxidative stress. Immunomodulatory agents have provided new therapeutic options. Natalizumab (Tysabri, Biogen Idec, Inc. and Elan Pharmaceuticals, Inc.; a monoclonal antibody directed against alpha-4-beta-1-integrin) is the selective adhesion inhibitor approved for treatment of relapsing-remitting-MS (RR-MS). Thus, the present work evaluates the effects of natalizumab on Nrf2 concentrations and oxidative damage levels in patients with RR-MS. Twenty-two patients with RR-MS treated with natalizumab were included in this work. Patients fulfilled the revised McDonald criteria and were scheduled to star treatment with 300 mg natalizumab intravenously once monthly (infusion every 4 weeks), for 14 months, according to Spanish guidelines. Carbonylated proteins, 8-hydroxy-2’-deoxyguanosine (8OHdG) and Nrf2 levels were measured at baseline and following treatment. Treatment with natalizumab caused a significant effect on Nrf2 expression and translocation, increasing in nuclei and cytoplasms in the white blood cells (WBC) of patients with RR-MS. This phenomenon was associated with a reduction in oxidative damage. This data suggest that natalizumab the cytoplasm and nucleus levels of WBC in patients with RR-MS.

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The role of iNOS in colon tumor viability GRACIELE ALMEIDA AND

OLIVEIRA, TATIANA ALVAREZ RINALDI, HUGO PEQUENO MONTEIRO DE

Laboratório de Sinalização Celular, Centro de Terapia Celular e Molecular, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil

The regulation of cell proliferation and longevity through cell signaling pathway is important to cellular homeostasis. Aberrations in this regulation are associated with tumor development. Nitric Oxide (NO) is a molecule permeable to cell lipid layer working as a messenger in intercellular communication and intracellular signaling. In mammalian, NO synthase enzymes (NOS) are responsible by NO biosynthesis. Three NOS isoform are well known: neuronal (nNOS), endothelial (eNOS) and inducible NOS (iNOS). Caloric restriction has been widely employed as a protocol to increases cell viability and longevity (1) and nutrients decrease without mal-nutrition were showed to promote eNOS expression (2,3). The aim of this study is to investigate the regulation of iNOS expression in metastatic human colon cancer cell line SW 620 maintained in restricted levels of nutrients. We will also investigate NO production, cell proliferation, and viability. We observed that cells cultured in absence or low concentrations of fetal bovine serum (FBS) exhibit increased iNOS protein expression and NO production. Cell proliferation decreases as compared to cells growing in the presence of 10% FBS. Furthermore, these cells are more viable. To test the importance of iNOS in this process we silence the expression of iNOS through permanent transfection of interference RNA. As opposed to parental SW 620 cells, iNOS silenced cells have no difference in NO generation and viability when growing in absence or presence of FBS (10%). Altogether, these results suggest that NO and iNOS play an important role in metastatic tumor cell culture viability. 1. Barros, M.H., da Cunha, F.M., Oliveira, G.A., Tahara, E.B., Kowaltowski, A.J. (2010). Mech Ageing Dev. 131:494-502. 2. Cerqueira, F.M. Laurindo, F.R., Kowaltowski, A.J. (2011) PLoS One. 6:18433. 3. Nisoli, E. et al. (2005) Science. 310:314-7.

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Anthocyanin metabolites improve human adipocyte insulin sensitivity by activating Nrf2/PPARgamma signalling pathway GIADA AMBROSINI, MASSIMO D’ARCHIVIO, BEATRICE SCAZZOCCHIO, ROSARIA VARÌ, CARMELINA FILESI, CARMELA SANTANGELO, CLAUDIO GIOVANNINI, AND ROBERTA MASELLA Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Roma, Italy

Anthocyanins naturally occurring in vegetables, fruits and beverages appear to exert beneficial effects in preventing oxidative stressassociated diseases, such as type 2 diabetes and insulin resistance, likely because of their anti-oxidant/anti-inflammatory properties. In particular, anthocyanins have been demonstrated to ameliorate hyperglycemia and insulin sensitivity. We have recently demonstrated that the main anthocyanin metabolite, the protocatechuic acid (PCA), exerts insulin-like activities in human visceral adipocytes by increasing PPARγ expression and activation, as well as adiponectin secretion. Aim of the study was to investigate the molecular mechanisms responsible for such activation. In particular we evaluated the involvement of the transcription factor Nrf2. Human adipocytes were isolated from visceral adipose tissue biopsies. In cells treated with 25μM PCA, the nuclear translocation of Nrf2, and adiponectin and PPARγ activation were evaluated by immunoblotting. To define the role of Nrf2 in modulating PCA effects, small-interfering RNA (siRNA) technique was used to inhibit transcription factor activity. In a set of experiments aimed at defining the specific signalling kinase involved in Nrf2 activation, the cells were treated with specific kinase inhibitors before PCA addition. We found an increase in Nrf2 nuclear level (p