Inflammation, stress oxydant, profil métabolique : influence des apports alimentaires et de la dépense énergétique

Le risque cardiométabolique (RCM) représente l’ensemble de tous les facteurs de risque pour les maladies cardiovasculaires et le diabète de type 2, incluant les facteurs de risque traditionnels et ceux émergents. Les évidences indiquent que la résistance à l’insuline, l’inflammation et le stress oxydant jouent un rôle clé dans le RCM, bien que l’acteur initiateur des altérations métaboliques caractéristiques du RCM reste encore à définir. Les femmes post-ménopausées constituent un sous-groupe important de la population puisque le risque de complications cardiométaboliques augmente après la ménopause. Les facteurs de RCM peuvent être modulés par l’alimentation, l’activité physique et la perte de poids. Alors que l’étude de nutriments / aliments spécifiques a permis de mieux comprendre l’implication de l’alimentation dans le RCM, celle de la qualité de l’alimentation est prometteuse. L’activité physique a des effets bénéfiques sur le RCM bien démontrés chez des personnes actives. Cependant, la relation entre la dépense énergétique et le RCM chez des individus sédentaires a été moins investiguée. De même, peu ou pas de données existent quant à une interaction synergique possible entre l’alimentation et l’activité physique sur le RCM. L’objectif de la présente thèse est d’investiguer les relations entre l’alimentation, l’activité physique, le stress oxydant et le RCM chez des femmes post-ménopausées en surpoids ou obèses, sédentaires et sans autres complications métaboliques. Les résultats montrent que d’une part, chez ces femmes sédentaires, une dépense énergétique active (DÉAP) élevée est associée à un meilleur profil inflammatoire, indépendamment de l’adiposité. D’autre part, il existe une relation synergique entre la qualité alimentaire et la DÉAP associée à un meilleur RCM. Une qualité alimentaire élevée combinée à une DÉAP élevée est associée à un meilleur profil lipidique et lipoprotéique et à une inflammation sub-clinique moindre, indépendamment de l’adiposité. Par ailleurs, dans une étude pilote, seuls des effets indépendants des changements de la qualité alimentaire et de la DÉAP sur les changements dans les facteurs de RCM ont été observés suite à cette diète hypocalorique de 6 mois, indépendamment du changement de l’adiposité encouru. En effet, au-delà de la réduction de l’adiposité et de l’amélioration du profil lipoprotéique induites par l’intervention, l’amélioration de la qualité alimentaire et de la DÉAP est associée, indépendamment l’une de l’autre, à une meilleure pression artérielle et un meilleur profil lipidique. Par ailleurs, une modification du système glutathion, un des systèmes antioxydants les plus communs de l’organisme, est associée à un RCM élevé. Une activité élevée de la glutathion peroxydase est associée à une résistance à l’insuline et à une épaisseur plus importante de l’intima-media de la carotide. Ces relations pourraient être médiées par un stress réducteur. En conclusion, l’adoption d’une saine alimentation et la pratique d’activités physiques doivent être encouragées dans les interventions visant à contrer l’obésité et ses complications, même en absence d’un changement d’adiposité. D’autre part, l’activité de la glutathion peroxydase pourrait être un paramètre impliqué dans le développement de désordres cardiométaboliques sub-cliniques et asymptomatiques chez des femmes obèses. D’autres investigations sont requises pour confirmer ces observations et élucider les mécanismes d’action impliqués.

Zea mays annexins modulate cytosolic free Ca2+ and generate a Ca2+-permeable conductance

Regulation of reactive oxygen species and cytosolic free calcium ([Ca2+](cyt)) is central to plant function. Annexins are small proteins capable of Ca2+-dependent membrane binding or membrane insertion. They possess structural motifs that could support both peroxidase activity and calcium transport. Here, a Zea mays annexin preparation caused increases in [Ca2+] cyt when added to protoplasts of Arabidopsis thaliana roots expressing aequorin. The pharmacological profile was consistent with annexin activation (at the extracellular plasma membrane face) of Arabidopsis Ca2+-permeable nonselective cation channels. Secreted annexins could therefore modulate Ca2+ influx. As maize annexins occur in the cytosol and plasma membrane, they were incorporated at the intracellular face of lipid bilayers designed to mimic the plasma membrane. Here, they generated an instantaneously activating Ca2+-permeable conductance at mildly acidic pH that was sensitive to verapamil and Gd3+ and had a Ca2+-to-K+ permeability ratio of 0.36. These results suggest that cytosolic annexins create a Ca2+ influx pathway directly, particularly during stress responses involving acidosis. A maize annexin preparation also demonstrated in vitro peroxidase activity that appeared independent of heme association. In conclusion, this study has demonstrated that plant annexins create Ca2+-permeable transport pathways, regulate [Ca2+] cyt, and may function as peroxidases in vitro.

Interaction of heat-moisture conditions and physical properties in oat processing: II. Flake quality

Product quality is an important determinant of consumer acceptance. Consistent oat flake properties are thus necessary in the mill as well as in the marketplace. The effects of kilning and tempering conditions (30, 60 or 90 min at 80, 95 or 110 degrees C) on flake peroxidase activity, size, thickness, strength and water absorption were therefore determined. After kilning, some peroxidase activity remained but steaming and tempering effectively destroyed the activity of these enzymes. Thus the supposed protective effect of kilning or groat durability was not confirmed. Kilning resulted in an increase in flake specific weight, but no other significant effect on flake quality was observed. Tempering time and temperature interacted significantly to produce complex effects on flake specific weight, thickness and water absorption. Flake thickness and specific weight were significantly correlated (r = 0.808, n = 54). Longer tempering times resulted in an increased fines' fraction, from 1.45% at 30 min to 1.75% at 90 min. It is concluded that whilst kilning has little effect on flake quality, the heat treatment immediately prior to flaking, can be used to adjust flake quality independently of flake thickness.

Supranutritional selenium induces alterations in molecular targets related to energy metabolism in skeletal muscle and visceral adipose tissue of pigs

While selenium (Se) is an essential micronutrient for humans, epidemiological studies have raised concern that supranutritional Se intake may increase the risk to develop Type 2 diabetes mellitus (T2DM). We aimed to determine the impact of Se at a dose and source frequently ingested by humans on markers of insulin sensitivity and signalling. Male pigs were fed either a Se-adequate (0.17 mg Se/kg) or a Se-supranutritional (0.50 mg Se/kg; high-Se) diet. After 16 weeks of intervention, fasting plasma insulin and cholesterol levels were non-significantly increased in the high-Se pigs, whereas fasting glucose concentrations did not differ between the two groups. In skeletal muscle of high-Se pigs, glutathione peroxidase activity was increased, gene expression of forkhead box O1 transcription factor and peroxisomal proliferator-activated receptor- coactivator 1 were increased and gene expression of the glycolytic enzyme pyruvate kinase was decreased. In visceral adipose tissue of high-Se pigs, mRNA levels of sterol regulatory element-binding transcription factor 1 were increased, and the phosphorylation of Akt, AMP-activated kinase and mitogen-activated protein kinases was affected. In conclusion, dietary Se oversupply may affect expression and activity of proteins involved in energy metabolism in major insulin target tissues, though this is probably not sufficient to induce diabetes.

Thiol and Sulfenic Acid Oxidation of AhpE, the One-Cysteine Peroxiredoxin from Mycobacterium tuberculosis: Kinetics, Acidity Constants, and Conformational Dynamics

Drug resistance and virulence of Mycobacterium tuberculosis are partially related to the pathogen`s antioxidant systems. Peroxide detoxification in this bacterium is achieved by the heme-containing catalase peroxidase and different two-cysteine peroxiredoxins. M. tuberculosis genome also codifies for a putative one-cysteine peroxiredoxin, alkyl hydroperoxide reductase E (MtAhpE). Its expression was previously demonstrated at a transcriptional level, and the crystallographic structure of the recombinant protein was resolved under reduced and oxidized states. Herein, we report that the conformation of MtAhpE changed depending on its single cysteine redox state, as reflected by different tryptophan fluorescence properties and changes in quaternary structure. Dynamics of fluorescence changes, complemented by competition kinetic assays, were used to perform protein functional studies. MtAhE reduced peroxynitrite 2 orders of magnitude faster than hydrogen peroxide (1.9 x 10(7) M(-1) s(-1) vs 8.2 x 10(4) M(-1) s(-1) at pH 7.4 and 25 degrees C, respectively). The latter also caused cysteine overoxidation to sulfinic acid, but at much slower rate constant (40 M(-1) s(-1)). The pK(a) of the thiol in the reduced enzyme was 5.2, more than one unit lower than that of the sulfenic acid in the oxidized enzyme. The pH profile of hydrogen peroxide-mediated thiol and sulfenic acid oxidations indicated thiolate and sulfenate as the reacting species. The formation of sulfenic acid as well as the catalytic peroxidase activity of MtAhpE was demonstrated using the artificial reducing substrate thionitrobenzoate. Taken together, our results indicate that MtAhpE is a relevant component in the antioxidant repertoire of M. tuberculosis probably involved in peroxide and specially peroxynitrite detoxification.

Hypermethioninemia provokes oxidative damage and histological changes in liver of rats

In the present study we evaluated the effect of chronic methionine administration on oxidative stress and biochemical parameters in liver and serum of rats, respectively. We also performed histological analysis in liver. Results showed that hypermethioninemia increased chemiluminescence, carbonyl content and glutathione peroxidase activity, decreased total antioxidant potential, as well as altered catalase activity. Hypermethioninemia increased synthesis and concentration of glycogen, besides histological studies showed morphological alterations and reduction in the glycogen/glycoprotein content in liver. Serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and glucose were increased in hypermethioninemic rats. These findings suggest that oxidative damage and histological changes caused by methionine may be related to the hepatic injury observed in hypermethioninemia. (C) 2009 Elsevier Masson SAS. All rights reserved.

Differential gene expression analysis of iodide-treated rat thyroid follicular cell line PCCl3

The inhibitory effect of supraphysiological iodide concentrations on thyroid hormone synthesis (Wolff - Chaikoff effect) and on thyrocyte proliferation is largely known as iodine autoregulation. However, the molecular mechanisms by which iodide modulates thyroid function remain unclear. In this paper, we analyze the transcriptome profile of the rat follicular cell lineage PCCl3 under untreated and treated conditions with 10 (- 3) M sodium iodide (NaI). Serial analysis of gene expression (SAGE) revealed 84 transcripts differentially expressed in response to iodide (p <= 0.001). We also showed that iodide excess inhibits the expression of essential genes for thyroid differentiation: Tshr, Nis, Tg, and Tpo. Relative expression of 14 of 20 transcripts selected by SAGE was confirmed by real-time PCR. Considering the key role of iodide organification in thyroid physiology, we also observed that both the oxidized form of iodide and iodide per se are responsible for gene expression modulation in response to iodide excess. (c) 2008 Elsevier Inc. All rights reserved.

PAS-1, a protein from Ascaris suum, modulates allergic inflammation via IL-10 and IFN-gamma, but not IL-12

Helminths and their products have a profound immunomodulatory effect upon the inductive and effector phases of inflammatory responses, including allergy. We have demonstrated that PAS-1, a protein isolated from Ascaris strum worms, has an inhibitory effect on lung allergic inflammation due to its ability to down-regulate eosinophilic inflammation, Th2 cytokine release and IgE antibody production. Here, we investigated the role of IL-12, IFN-gamma and IL-10 in the PAS-1-induced inhibitory mechanism using a murine model of asthma. Wild type C57BL/6, IL-12(-/-), IFN-gamma(-/-) and IL-10(-/-) mice were immunized with PAS-1 and/or OVA and challenged with the same antigens intranasally. The suppressive effect of PAS-I was demonstrated on the cellular influx into airways, with reduction of eosinophil number and eosinophil peroxidase activity in OVA + PAS-1-immunized wild type mice. This effect well correlated with a significant reduction in the levels of IL-4, IL-5, IL-13 and eotaxin in BAL fluid. Levels of IgE and IgG1 antibodies were also impaired in serum from these mice. The inhibitory activity of PAS-I was also observed in IL-12(-/-) mice, but not in IFN-gamma(-/-) and IL-10(-/-) animals. These data show that IFN-gamma and IL-10, but not IL-12, play an important role in the PAS-1 modulatory effect. (C) 2008 Elsevier Ltd. All rights reserved.

Superoxide radical protects liposome-contained cytochrome c against oxidative damage promoted by peroxynitrite and free radicals

The effects of nitrosative species on cyt c structure and peroxidase activity were investigated here in the presence of O(2)(center dot-) and anionic and zwitterionic vesicles. Nitrosative species were generated by 3-morpholinesydnonymine (SIN1) decomposition, using cyt c heme iron and/or molecular oxygen as electron acceptor. Far-and near-UV CD spectra of SIN1-treated cyt c revealed respectively a slight decrease of a-helix content (from 39 to 34%) and changes in the tryptophan structure accompanied by increased fluorescence. The Soret CD spectra displayed a significant decrease of the positive signal at 403 nm. EPR spectra revealed the presence of a low-spin cyt c form (S = 1/2) with g(1) = 2.736, g(2) = 2.465, and g(3) = 2.058 after incubation with SIN1. These data suggest that the concomitant presence of NO(center dot) and O(2)(center dot-) generated from dissolved oxygen, in a system containing cyt c and liposomes, promotes chemical and conformational modi. cations in cyt c, resulting in a hypothetical bis-histidine hexacoordinated heme iron. We also show that, paradoxically, O(2)(center dot-) prevents not only membrane lipoperoxidation by peroxide-derived radicals but also oxidation of cyt c itself due to the ability of O(2)(center dot-) to reduce heme iron. Finally, lipoperoxidation measurements showed that, although it is a more efficient peroxidase, SIN1-treated cyt c is not more effective than native cyt c in promoting damage to anionic liposomes in the presence of tert-ButylOOH, probably due to loss of affinity with negatively charged lipids. (C) 2009 Elsevier Inc. All rights reserved.

Superoxide Dismutase 1-mediated Production of Ethanol- and DNA-derived Radicals in Yeasts Challenged with Hydrogen Peroxide MOLECULAR INSIGHTS INTO THE GENOME INSTABILITY OF PEROXIREDOXIN-NULL STRAINS

Peroxiredoxins are receiving increasing attention as defenders against oxidative damage and sensors of hydrogen peroxide-mediated signaling events. In the yeast Saccharomyces cerevisiae, deletion of one or more isoforms of the peroxiredoxins is not lethal but compromises genome stability by mechanisms that remain under scrutiny. Here, we show that cytosolic peroxiredoxin-null cells (tsa1 Delta tsa2 Delta) are more resistant to hydrogen peroxide than wildtype (WT) cells and consume it faster under fermentative conditions. Also, tsa1 Delta tsa2 Delta cells produced higher yields of the 1-hydroxyethyl radical from oxidation of the glucose metabolite ethanol, as proved by spin-trapping experiments. A major role for Fenton chemistry in radical formation was excluded by comparing WT and tsa1 Delta tsa2 Delta cells with respect to their levels of total and chelatable metal ions and of radical produced in the presence of chelators. The main route for 1-hydroxyethyl radical formation was ascribed to the peroxidase activity of Cu, Zn-superoxide dismutase (Sod1), whose expression and activity increased similar to 5- and 2-fold, respectively, in tsa1 Delta tsa2 Delta compared with WT cells. Accordingly, overexpression of human Sod1 in WT yeasts led to increased 1-hydroxyethyl radical production. Relevantly, tsa1 Delta tsa2 Delta cells challenged with hydrogen peroxide contained higher levels of DNA-derived radicals and adducts as monitored by immuno-spin trapping and incorporation of (14)C from glucose into DNA, respectively. The results indicate that part of hydrogen peroxide consumption by tsa1 Delta tsa2 Delta cells is mediated by induced Sod1, which oxidizes ethanol to the 1-hydroxyethyl radical, which, in turn, leads to increased DNA damage. Overall, our studies provide a pathway to account for the hypermutability of peroxiredoxin-null strains.

Increased SOD1 association with chromatin, DNA damage, p53 activation, and apoptosis in a cellular model of SOD1-linked ALS

Mutations in the gene encoding cytosolic Cu,Zn-superoxide dismutase (SOD1) have been linked to familial amyotrophic lateral sclerosis (FALS). However the molecular mechanisms of motor neuron death are multifactorial and remain unclear. Here we examined DNA damage;p53 activity and apoptosis in SH-SY5Y human neuroblastoma cells transfected to achieve low-level expression of either wild-type or mutant Gly(93) --> Ala (G93A) SOD1, typical of FALS. DNA damage was investigated by evaluating the levels of 8-oxo-7,8-dihydro-2`-deoxyguanosine (8-oxodGuo) and DNA strand breaks. Significantly higher levels of DNA damage, increased p53 activity, and a greater percentage of apoptotic cells were observed in SH-SY5Y cells transfected with G93A SOD1 when compared to cells overexpressing wild-type SOD1 and untransfected cells. Western blot, FACS, and confocal microscopy analysis demonstrated that G93A SOD1 is present in the nucleus in association with DNA. Nuclear G93A SOD1 has identical superoxide dismutase activity but displays increased peroxidase activity when compared to wild-type SOD1. These results indicate that the G93A mutant SOD1 association with DNA might induce DNA damage and trigger the apoptotic response by activating p53. This toxic activity of mutant SOD1 in the nucleus may play an important role in the complex mechanisms associated with motor neuron death observed in ALS pathogenesis. (C) 2010 Elsevier B.V. All rights reserved.

Antioxidant defences and homeostasis of reactive oxygen species in different human mitochondrial DNA-depleted cell lines

Three pairs of parental (ρ+) and established mitochondrial DNA depleted (ρ0) cells, derived from bone, lung and muscle were used to verify the influence of the nuclear background and the lack of efficient mitochondrial respiratory chain on antioxidant defences and homeostasis of intracellular reactive oxygen species (ROS). Mitochondrial DNA depletion significantly lowered glutathione reductase activity, glutathione (GSH) content, and consistently altered the GSH2 : oxidized glutathione ratio in all of the ρ0 cell lines, albeit to differing extents, indicating the most oxidized redox state in bone ρ0 cells. Activity, as well as gene expression and protein content, of superoxide dismutase showed a decrease in bone and muscle ρ0 cell lines but not in lung ρ0 cells. GSH peroxidase activity was four times higher in all three ρ0 cell lines in comparison to the parental ρ+, suggesting that this may be a necessary adaptation for survival without a functional respiratory chain. Taken together, these data suggest that the lack of respiratory chain prompts the cells to reduce their need for antioxidant defences in a tissue-specific manner, exposing them to a major risk of oxidative injury. In fact bone-derived ρ0 cells displayed the highest steady-state level of intracellular ROS (measured directly by 2',7'-dichlorofluorescin, or indirectly by aconitase activity) compared to all the other ρ+ and ρ0 cells, both in the presence or absence of glucose. Analysis of mitochondrial and cytosolic/iron regulatory protein-1 aconitase indicated that most ROS of bone ρ0 cells originate from sources other than mitochondria.

Morphologic and functional characterization of granulocytes and macrophages in embryonic and adult zebrafish.

The zebrafish is a useful model organism for developmental and genetic studies. The morphology and function of zebrafish myeloid cells were characterized. Adult zebrafish contain 2 distinct granulocytes, a heterophil and a rarer eosinophil, both of which circulate and are generated in the kidney, the adult hematopoietic organ. Heterophils show strong histochemical myeloperoxidasic activity, although weaker peroxidase activity was observed under some conditions in eosinophils and erythrocytes. Embryonic zebrafish have circulating immature heterophils by 48 hours after fertilization (hpf). A zebrafish myeloperoxidase homologue (myeloid-specific peroxidase; mpx) was isolated. Phylogenetic analysis suggested it represented a gene ancestral to the mammalian myeloperoxidase gene family. It was expressed in adult granulocytes and in embryos from 18 hpf, first diffusely in the axial intermediate cell mass and then discretely in a dispersed cell population. Comparison of hemoglobinized cell distribution, mpx gene expression, and myeloperoxidase histochemistry in wild-type and mutant embryos confirmed that the latter reliably identified a population of myeloid cells. Studies in embryos after tail transection demonstrated that mpx- and peroxidase-expressing cells were mobile and localized to a site of inflammation, indicating functional capability of these embryonic granulocytes. Embryonic macrophages removed carbon particles from the circulation by phagocytosis. Collectively, these observations have demonstrated the early onset of zebrafish granulopoiesis, have proved that granulocytes circulate by 48 hpf, and have demonstrated the functional activity of embryonic granulocytes and macrophages. These observations will facilitate the application of this genetically tractable organism to the study of myelopoiesis.

The effect of short-term canola oil ingestion on oxidative stress in the vasculature of stroke-prone spontaneously hypertensive rats

Background: This study aimed to determine if 25 days of canola oil intake in the absence of excess dietary salt or together with salt loading affects antioxidant and oxidative stress markers in the circulation. A further aim was to determine the mRNA expression of NADPH oxidase subunits and superoxide dismutase (SOD) isoforms in the aorta of stroke-prone spontaneously hypertensive (SHRSP) rats.

Methods: Male SHRSP rats, were fed a defatted control diet containing 10% wt/wt soybean oil or a defatted treatment diet containing 10% wt/wt canola oil, and given tap water or water containing 1% NaCl. Blood was collected at the end of study for analysis of red blood cell (RBC) antioxidant enzymes, RBC and plasma malondialdehyde (MDA), plasma 8-isoprostane and plasma lipids. The aorta was removed and the mRNA expression of NOX2, p22phox, CuZn-SOD, Mn-SOD and EC-SOD were determined.

Results: In the absence of salt, canola oil reduced RBC SOD and glutathione peroxidase, and increased total cholesterol and LDL cholesterol compared with soybean oil. RBC glutathione peroxidase activity was significantly lower in both the salt loaded groups compared to the soybean oil only group. In addition, RBC MDA and plasma HDL cholesterol were significantly higher in both the salt loaded groups compared to the no salt groups. Plasma MDA concentration was higher and LDL cholesterol concentration lower in the canola oil group loaded with salt compared to the canola oil group without salt. The mRNA expression of NADPH oxidase subunits and SOD isoforms were significantly reduced in the canola oil group with salt compared to canola oil group without salt.

Conclusion: In conclusion, these results indicate that canola oil reduces antioxidant status and increases plasma lipids, which are risk factors for cardiovascular disease. However, canola oil in combination with salt intake increased MDA, a marker of lipid peroxidation and decreased NAPDH oxidase subunits and aortic SOD gene expression.

Salt loading in canola oil fed SHRSP rats induces endothelial dysfunction

This study aimed to determine if 50 days of canola oil intake in the absence or presence of salt loading affects: (1) antioxidant and oxidative stress markers, (2) aortic mRNA of NADPH oxidase (NOX) subunits and superoxide dismutase (SOD) isoforms and (3) endothelial function in SHRSP rats. SHRSP rats were fed a diet containing 10 wt/wt% soybean oil or 10 wt/wt% canola oil, and given tap water or water containing 1% NaCl for 50 days. Without salt, canola oil significantly increased RBC SOD, plasma cholesterol and triglycerides, aortic p22phox, NOX2 and CuZn-SOD mRNA, and decreased RBC glutathione peroxidase activity. With salt, canola oil reduced RBC SOD and catalase activity, LDL-C, and p22phox mRNA compared with canola oil alone, whereas plasma malondialdehyde (MDA) was reduced and RBC MDA and LDL-C were higher. With salt, the canola oil group had significantly reduced endothelium-dependent vasodilating responses to ACh and contractile responses to norepinephrine compared with the canola oil group without salt and to the WKY rats. These results indicate that ingestion of canola oil increases O2 - generation, and that canola oil ingestion in combination with salt leads to endothelial dysfunction in the SHRSP model.