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Evidence is mounting of the involvement of mitochondrial dysfunction in insulin resistance, diabetes and associated complications. This review aims to provide an overview of the effects of insulin resistance on mitochondrial function in several tissues. We consider the pathogenesis of insulin resistance from a mitochondrial perspective and contemplate potential beneficial effects of strategies aimed at modulating mitochondrial function in insulin resistance, including insulin and insulin-sensitizing drugs, antioxidants, and selectively targeting antioxidants to mitochondria.

A variety of nanoparticles have been proposed for several biomedical applications. To gauge the therapeutic potential of these nanoparticles, in vivo biodistribution is essential and mandatory. In the present study, ceria nanoparticles (5 nm average particle size) were labeled with F-18 to study their in vivo biodistribution in rats by positron emission tomography (PET). The F-18 isotope was anchored by reaction of N-succinimidyl 4-[F-18]fluorobenzoate (F-18-SFB) with a modified nanoparticle surface obtained by silylation with 3-aminopropylsilyl. Radiolabeled ceria nanoparticles accumulated mainly in lungs, spleen, and liver. Metabolic products of the radiolabeled nanoparticulate material were excreted into the urinary tract., The present work was supported by the Spanish MICINN (Grants CTQ-2009-11586, CTQ2006-06785, and CTQ2007-67805-AR07, PI10/1195, AP192/11); the Fondo de Investigacion Sanitaria (FIS) of the Instituto de Salud Carlos III (Grants PS09/02620, PI10/1195, and PS09/02217), the Generalitat Valenciana (Grant ACOMP/2012/045), La Marato Fundation (Grant 090530), and by CDTI under the CENIT Programme (AMIT Project) and the Spanish Ministry of Science and Innovation. V.M.V. is a recipient of a contract from the Regional Ministry of Health of the Valencian Regional Government and Carlos III Health Institute (CES10/030).

Inhaled radioactive CO is currently the tracer of choice for blood volume quantification by positron emission tomography (PET). This measurement is of great interest for several clinical and research applications. However, owing to the short half-life of the radiolabeled CO, it can only be used in centers equipped with a cyclotron. In the present work, we propose an alternative method to label the red blood cells with [(18) F] in order to obtain blood volume measurements by PET. The use of the radioactive synthon [(18) F] N-succinimidyl 4-[(18) F]fluorobenzoate ([(18) F]SFB) was evaluated for erythrocyte labeling and PET blood volume imaging. The images provided by [(18) F]SFB labeled erythrocytes were compared with those obtained with inhaled [(11) C]CO. Blood volumes obtained with [(18) F]SFB labeled erythrocytes were similar to those obtained with [(11) C]CO in all of the evaluated organs with the exception of spleen, which presented lower uptake with this method. Since the [(18) F]-SFB binds irreversibly to red blood cells, in vivo stability of the radiolabel was higher compared with the [(11) C]CO method. Additionally, owing to the longer half-life and the shorter positron range of [(18) F], the image quality was also higher with the [(18) F]SFB radiolabeled erythrocytes. The labeling of red blood with [(18) F]SFB represents an advantageous alternative to radioactive CO for blood volume measurement by PET and cardiovascular isotopic imaging.

Resumen Introduccion La combinacion de farmacos es habitual en el tratamiento de las dislipidemias, siendo las estatinas y la ezetimiba la asociacion mas utilizada. Al actuar a distinto nivel, con esta combinacion se facilita la consecucion de los objetivos terapeuticos. Sin embargo, poco se sabe sobre como estos farmacos –en monoterapia o asociados– podrian afectar cualitativamente la composicion de las subfracciones lipoproteicas, las cuales difieren en tamano y potencial aterogenico, asi como su efecto antiinflamatorio. Metodos Se incluyeron 39 pacientes con hiperlipidemia, los cuales fueron aleatoriamente distribuidos en 2 grupos: a uno se le administro simvastatina (40 mg/dia) y al otro ezetimiba (10 mg/dia) durante 4 semanas, transcurridas las cuales se les adminsitro de forma conjunta ambos farmacos durante 4 semanas mas. Se valoro el perfil lipidico, subfracciones lipoproteicas de LDL y HDL, asi como parametros inflamatorios. Resultados El tratamiento farmacologico en monoterapia (simvastatina vs ezetimiba) redujo el cLDL (−40,2 vs. −19,6%) y el colesterol no-HDL (−37,1 vs. −18,8%) y la simvastatina en monoterapia indujo un descenso de trigliceridos (−18,6%) y un aumento de cHDL (+7,4%). La coadministracion produjo una reduccion de hasta el 57% sobre el cLDL. La terapia con simvastatina disminuyo las subfracciones de HDL mas pequenas y aumento las mas grandes. Ademas, aumento significativamente el diametro, reduciendo el porcentaje de sdLDL y provoco un descenso significativo del TNFα. Conclusiones La combinacion de simvastatina y ezetimiba mostro un efecto sumatorio hipocolesterolemiante. Asimismo, la simvastatina redujo las subfracciones lipoproteicas mas aterogenicas y mas a largo plazo mostro efectos antiinflamatorios.

The GABA-ergic system, known to regulate neural tissue genesis during cortical development, has been postulated to play a role in cerebral aging processes. Using in vivo molecular imaging and voxel-wise quantification, we aimed to assess the effects of aging on the benzodiazepine (BDZ) recognition site of the GABA A receptor. To visualize BDZ site availability, [C-11]-flumazenil microPET acquisitions were conducted in young and old rats. The data were analyzed and region of interest analyses were applied to validate the voxel-wise approach. We observed decreased [C-11]-flumazenil binding in the aged rat brains in comparison with the young control group. More specifically, clusters of reduced radioligand uptake were detected in the bilateral hippocampus, cerebellum, midbrain, and bilateral frontal and parieto-occipital cortex. Our results support the pertinence of voxel-wise quantification in the analysis of microPET data. Moreover, these findings indicate that the aging process involves declines in neural BDZ recognition site availability, proposed to reflect alterations in GABA A receptor subunit polypeptide expression. (C) 2012 Elsevier Inc. All rights reserved.

The medical use of nitroglycerin (GTN) is limited by patient tolerance. The present study evaluated the role of mitochondrial Complex I in GIN biotransformation and the therapeutic effect of mitochondrial antioxidants. The development of GIN tolerance (in rat and human vessels) produced a decrease in mitochondrial 02 consumption. Co-incubation with the mitochondria-targeted antioxidant mitoquinone (MQ 10(-6) mol/L) or with glutathione ester (GEE, 10(-4) mol/L) blocked GTN tolerance and the effects of GTN on mitochondrial respiration and aldehyde dehydrogenase 2 (ALDH-2) activity. Biotransformation of GTN depended on the mitochondria being functionally active, particularly mitochondrial Complex I. Tolerance induced mitochondrial ROS production and oxidative stress, though these effects were not detected in HUVEC rho(0) cells or Complex I mutant cells. Experiments performed to evaluate Complex I-dependent respiration demonstrated that its inhibition by GIN was prevented by the antioxidants in control samples. These results point to a key role for mitochondrial Complex I in the adequate functioning of ALDH-2. In addition, we have identified mitochondrial Complex I as one of the targets at which the initial oxidative stress responsible for GIN tolerance takes place. Our data also suggest a role for mitochondrial-antioxidants as therapeutic tools in the control of the tolerance that accompanies chronic nitrate use. (C) 2012 Elsevier B.V. All rights reserved.

Introduction Erectile dysfunction (ED) is associated with cardiovascular disease (CVD) because it is highly prevalent among those with cardiovascular risk factors (CVRFs). Moreover, it precedes the development of CVD and is considered a subrogate marker of subclinical CVD. Aim The aim of this study was to evaluate the presence of ED among patients with type 2 diabetes (DM2) without macroangiopathy, and to assess the association between ED and other CVRFs, chronic diabetes complications, silent myocardial ischemia (SMI), and peripheral arterial disease (PAD). Methods One hundred fifty‐four male patients with DM2 and without clinical evidence of CVD were included in the study. The presence of ED, PAD, SMI, chronic diabetic complications, and other CVRFs was evaluated in these patients. Main Outcome Measures PAD; SMI; ED; 24‐hour blood pressure Holter; lipid profile; insulin resistance; testosterone; chronic inflammation; nephropathy; retinopathy; neuropathy. Results Prevalence of ED was 68.2%. Patients with ED were older and characterized by DM2, systolic blood pressure (BP), retinopathy, and insulin treatment of longer duration than patients without ED, even when adjusting for age was performed. Adjusting for duration of diabetic condition revealed significant differences in age and systolic BP. Independent factors for ED were age (57.7 ± 7.5 years, relative risks [RR 1.1], P = 0.003) and duration of diabetes (9[3–15] years, RR 1.1, P = 0.006). SMI was detected in 13.6% of patients (18.1% in patients with ED vs. 4.1% in patients without ED). Asymptomatic PAD was detected in 13.2% of subjects (14.4% in patients with ED vs. 10.4% in patients without). Conclusions ED is highly prevalent in DM2, and is associated with the presence of SMI, higher systolic BP and chronic microvascular diabetic complications. Garcia‐Malpartida K, Marmol R, Jover A, Gomez‐Martinez MJ, Sola‐Izquierdo E, Victor VM, Rocha M, Sanmiguel D, and Hernandez‐Mijares A. Relationship between erectile dysfunction and silent myocardial ischemia in type 2 diabetic patients with no known macrovascular complications. J Sex Med 2011;8:2606–2616.

Phytosterols, which are structurally related to cholesterol, are found in all plant foods with highest concentration occurring in vegetable oils and nuts. Phytosterols are known to reduce serum low-density lipoprotein cholesterol level without changing high-density lipoprotein cholesterol or triglyceride levels. Daily consumption of phytosterols-enriched foods is widely used as a therapeutic option to lower plasma cholesterol and atherosclerotic disease risk. The cholesterol-lowering action of phytosterols is thought to occur, at least in part, through competitive replacement of dietary and biliary cholesterol in mixed micelles, which undermines the absorption of cholesterol. The aim of this review is to provide a general overview of available evidence regarding the effects of phytosterols on cholesterol metabolism and addressing issues related to efficacy as dose, length, frequency of consumption, type of phytosterol (sterols versus stanols) or food matrix. Furthermore, we will explore the factors that influence the response of individuals to phytosterol therapy and evaluate their safety and the possibility that elevated plasma phytosterol concentrations contribute to the development of premature coronary artery disease.

Diabetes is a severe, heterogeneous, multifactorial, chronic disease. Diabetes and oxidative stress are related to continuous and acute overproduction of reactive oxygen species (ROS). These ROS are released principally from mitochondria, but also have other sources. Oxidative stress seems to play an important role in mitochondria-mediated disease processes, though the exact molecular mechanisms responsible remain elusive. ROS are necessary for the proper functioning of the cell, but their excessive production can be harmful, making antioxidant defenses essential. Some substances with antioxidant properties, such as vitamins C and E, have been used to eradicate the oxidative stress associated with diabetes. The results of clinical trials employing anti-oxidative stress reagents in patients with diabetes are contradictory, perhaps due to inadequate study design or the specific targets selected. This review considers the process of diabetes from a mitochondrial perspective and evaluates strategies currently under development for the targeted delivery of antioxidants or other molecules to mitochondria. The evidence compiled herein endorses the selective targeting of specific molecules to mitochondria as an effective strategy for modulating mitochondrial respiration and ROS production and protecting mitochondria against oxidative stress.

Diabetes is associated with oxidative stress. This study evaluated the rates of oxidative stress and mitochondrial impairment in type 2 diabetes patients. The study population consisted of 182 diabetic patients and 50 body-composition- and age-matched controls. We assessed anthropometric and metabolic parameters and mitochondrial function by evaluating mitochondrial oxygen (O2) consumption, reactive oxygen species (ROS) production, glutathione (GSH) levels, GSH/GSSG ratio, mitochondrial membrane potential, and mitochondrial complex I activity in polymorphonuclear cells from diabetes type 2 patients. We found an increase in waist circumference and augmented serum levels of triglycerides, proinflammatory cytokines (IL-6 and TNF-α), homocysteine, glycated hemoglobin, ultrasensitive C-reactive protein, glucose, insulin, and homeostasis model assessment of insulin resistance score in diabetic patients versus controls. There was an impairment of mitochondrial function in diabetic patients, evidenced by a decrease in mitochondrial O2 consumption, an increase in ROS production, decreased GSH/GSSG ratio, a drop in GSH levels, and an undermining of the mitochondrial membrane potential. Furthermore, an impairment of mitochondrial complex I was detected. This study supports the hypothesis of an association of type 2 diabetes and the rate of impaired mitochondrial function. We also propose that one of the targets of oxidative stress responsible for diabetes is mitochondrial complex I.

Background/Objectives: The importance of both low-density lipoprotein cholesterol (LDLc) size and the apolipoprotein E (Apo E) in the atherogenic process is known, but there is little information with regard to the effect of phytosterols (PS) on these parameters. The aim of this study was to evaluate the influence of PS on lipid profile and LDLc size according to Apo E genotype. Subjects/Methods: This was a randomized parallel trial employing 75 mild-hypercholesterolemic subjects and consisting of two 3-month intervention phases. After 3 months of receiving a standard healthy diet, subjects were divided into two intervention groups: a diet group (n = 34) and a diet+PS group (n = 41) that received 2 g/day of PS. Total cholesterol (TC), triacylglycerols, LDLc, high-density lipoprotein cholesterol (HDLc), non-HDLc, Apo A-I and B-100, LDLc size and Apo E genotype were determined. Results: Patients receiving PS exhibited a significant decrease in TC (5.1%), LDLc (8.1%), non-HDLc (7.4%) and Apo B-100/Apo A-I ratio (7.7%), but these effects did not depend on Apo E genotype. No significant changes were found in lipid profile according to Apo E genotype when patients following dietary recommendations were considered as a whole population or separately. No variations in LDLc size were observed in any of the intervention groups. Conclusion: The results of this study show that Apo E genotype does not have an impact on the lipid response to PS as a cholesterol-lowering agent in mild-hypercholesterolemic patients. Furthermore, the evidence obtained confirms that LDLc particle size is not modified when PS are added to a standard healthy diet. European Journal of Clinical Nutrition (2011) 65, 255-261; doi:10.1038/ejcn.2010.241; published online 3 November 2010

Insulin resistance is a feature of polycystic ovary syndrome (PCOS) and is related to mitochondrial and endothelial function.We tested whether hyperandrogenic insulin-resistant women with PCOS, who have an increased risk of vascular disease, display impaired leukocyte-endothelium interactions, and mitochondrial dysfunction.This was a prospective controlled study conducted in an academic medical center.The study population consisted of 43 lean reproductive-age women with PCOS and 39 controls subjects.We evaluated anthropometric and metabolic parameters, adhesion molecules, and interactions between leukocytes and human umbilical vein endothelial cells. Mitochondrial function was studied by assessing mitochondrial oxygen consumption, membrane potential, reactive oxygen species production, glutathione levels (GSH), and the oxidized glutathione (GSSG)/GSH ratio in polymorphonuclear cells.Impairment of mitochondrial function was observed in the PCOS patients, evident in a decrease in oxygen consumption, an increase in reactive oxygen species production, a decrease in the GSH/GSSG ratio and GSH levels, and an undermining of the membrane potential. PCOS was related to a decrease in polymorphonuclear cell rolling velocity and an increase in rolling flux and adhesion. Increases in IL-6 and TNFα and adhesion molecules (vascular cell adhesion molecule-1 and E-selectin) were also observed.This study supports the hypothesis of an association between insulin resistance and an impaired endothelial and mitochondrial oxidative metabolism. The evidence obtained shows that the inflammatory state related to insulin resistance in PCOS induces a leukocyte-endothelium interaction. These findings may explain the increased risk of vascular disease in women with PCOS.

Mitochondrial dysfunction plays a key role in sepsis.We used a sepsis model of human endothelial cells (HUVEC) to study mitochondrial function during normoxic (21% O(2)) and hypoxic (1% O(2)) conditions.When stimulated with a LPS cocktail, HUVEC displayed an increase of nitric oxide (NO) in normoxic and hipoxic conditions, being higher at 21% O(2). LPS-activation for 24 h at 1% O(2) increased ROS production, which was reversed with the mitochondrial antioxidant Mitoquinone (MQ) and Glutathione Ethyl Ester (GEE). Activated cells displayed diminished mitochondrial O(2) consumption with specific inhibition of Complex I, accompanied by increase in tyrosine nitration and Type II NOS protein expression, effects which were recovered by antioxidants and/or with L-NAME. These parameters varied with O(2) environment, namely inhibition of respiration observed in both O(2) environments at 24 h was very similar, whereas O(2) consumption rate fell earlier in 1% O(2)-exposed cells. While no significant differences were detected at earlier time points, at 24 h tyrosine nitration was higher in normoxic vs. hypoxic cells.Mitochondria are heavily implicated in sepsis. Mitochondrial antioxidants provide a mechanistic model for the development of potential therapies.

[EN] Nanoparticles have been proposed for several biomedical applications; however, in vivo biodistribution studies to con¿rm their potential are scarce. Nanodiamonds are carbon nanoparticles that have been recently proposed as a promising biomaterial. In this study, we labeled nanodiamonds with 18F to study their in vivo biodistribution by positron emission tomography. Moreover, the impact on the biodistribution of their kinetic particle size and of the surfactant agents has been evaluated. Radiolabeled diamond nanoparticles accumulated mainly in the lung, spleen, and liver and were excreted into the urinary tract. The addition of surfactant agents did not lead to signi¿cant changes in this pattern, with the exception of a slight reduction in the urinary excretion rate. On the other hand, after ¿ltration of the radiolabeled diamond nanoparticles to remove those with a larger kinetic size, the uptake in the lung and spleen was completely inhibited and signi¿cantly reduced in the liver, The present work was supported by Spanish MICINN (Grant CTQ-2009-11586); the Fondo de Investigacion Sanitaria (FIS) of the Instituto de Salud Carlos III (Grants PS09/02620, PI10/1195, and PS09/02217), La Marato TV3 Foundation (Grant 090530), and CDTI under the CENIT Programme (AMIT Project) and supported by the Spanish Ministry of Science and Innovation. R.M. thanks the Spanish Ministry for a postgraduate scholarship. V.M.V. is a recipient of Fond de Investigacion Sanitaria (FIS) and Generalitat Valenciana contract (CES10/030)

Overproduction of reactive oxygen species (ROS) under pathophysiologic conditions is part of the disease process. These ROS are released from different sources, and in particular from mitochondria. Although the molecular mechanisms responsible for mitochondria-mediated disease processes are unclear, oxidative stress seems to play an important role. ROS are essential to cell function, but adequate levels of antioxidant defenses are required in order to avoid the harmful effects that excessive ROS production can produce. Mitochondrial oxidative stress damage and dysfunction contribute to a number of cell pathologies that manifest themselves through a range of conditions. The antioxidants available until now have not proved to be particularly effective against many of these disorders. It is possible that these antioxidants do not reach the sites of free radical generation, especially when mitochondria are the primary source of ROS. Recent developments in mitochondria-targeted antioxidants have moved closer to providing protection against mitochondrial oxidative damage. The SS (Szeto-Schiller) peptide antioxidants represent a novel approach that employs the targeted delivery of antioxidants to the inner mitochondrial membrane. These SS peptides scavenge hydrogen peroxide and peroxynitrite and inhibit lipid peroxidation. By reducing mitochondrial ROS, they inhibit mitochondrial permeability transition and cytochrome c release, thus preventing oxidant-induced cell death. Preclinical studies support the use of these peptides for ischemia-reperfusion injury and neurodegenerative disorders. Although peptides have often been considered to be poor drug candidates, the few that have been studied are promising agents for the treatment of diseases.

Gastrin release is affected by gastric inflammatory conditions. Antral G cells respond to inflammatory mediators by increasing gastrin secretion. Accumulating experimental evidence suggests that gastrin exerts immunomodulatory and proinflammatory effects. Gastrin could be a contributing factor to these pathologies, which may constitute a new justification for pharmacological blockade of gastrin action.

Phytosterols (PS) are recommended to reduce LDL-cholesterol. However, the influence of cholesterol and fat intake on the lipid-lowering effect of PS in mildly hypercholesterolaemia is unclear. Thus, the aim of the present study was to evaluate whether the efficacy of PS is related to the composition of saturated fat and dietary cholesterol intake. Additionally, serum carotenoid content was analysed to evaluate to what extent it was undermined by PS. This was a 3-month randomised, parallel trial with a three-arm design. Patients were divided into three groups: healthy diet (n 24), healthy diet + PS (n 31) and free diet + PS (n 29), receiving 2 g/d of PS. Healthy and free diets were characterised by a daily ingestion of 6.8% of saturated fat and 194.4 mg of cholesterol and 12.7% of saturated fat and 268.1 mg of cholesterol, respectively. After PS therapy, patients receiving the healthy diet + PS or a free diet + PS exhibited a similar reduction in total cholesterol (6.7 and 5.5 %), LDL-cholesterol (9.6 and 7.0 %), non-HDL-cholesterol (12.2 and 8.9%) and apo B-100/apo A-I ratio (11.5 and 11.6 %), respectively. In patients following the healthy diet, (beta-carotene concentration rose by 26.9 %, whereas the beta-carotene and lycopene levels dropped by 21.0 and 22.8% in the group receiving the free diet + PS, respectively. No change was observed in carotenoid levels in healthy diet + PS group. In conclusion, the efficacy of PS in relation to lipoprotein profile is not influenced by saturated fat or dietary cholesterol intake, which confirms the positive effect of healthy diet therapy in improving the negative effects that PS exert on carotenoid levels.

Functional impairment of endothelial activity (endothelial dysfunction) precedes the development of cardiovascular diseases (CVD). This condition is a result of a reduced bioavailability of nitric oxide (NO), a well known vasodilator, which is mainly due to increased NO degradation caused by its reaction with reactive oxygen species (ROS). Although there are several conditions that contribute independently to endothelial dysfunction, such as hyperglycemia, insulin resistance, hyperinsulinemia and dyslipidemia, increased oxidative stress seems to play a key role. In addition to their original pharmacological properties, drugs used clinically at present, including anti-hypertension reagents, angiotensin receptor blockers and anti-hyperlipidemic reagents such as statins, protect various organs via anti-oxidative stress mechanisms. Moreover, some substances with antioxidant properties, such as vitamin C or vitamin E, have been used to eradicate the oxidative stress associated with CVD. The results of the clinical trials employing anti-oxidative stress reagents in patients with CVD are contradictory, which could be a result of inadequate study design or selected targets. This review considers the process of endothelial dysfunction and CVD from a mitochondrial perspective and evaluates strategies currently under development for the targeted delivery of antioxidants or NO to mitochondria. It endorses the idea that selectively targeting specific antioxidants and NO donors to mitochondria is an effective strategy for modulating mitochondrial respiration and ROS production and protecting mitochondria against oxidative stress.

Efavirenz (EFV) is a non-nucleoside reverse transcriptase inhibitor (NNRTI) widely used in human immunodeficiency virus (HIV) infection therapy. It has been associated with hepatotoxic effects and alterations in lipid and body fat composition. Given the importance of the liver in lipid regulation, we have evaluated the effects of clinically used concentrations of EFV on the mitochondria and lipid metabolism of human hepatic cells in vitro. Mitochondrial function was rapidly undermined by EFV to an extent that varied with the concentration employed; in particular, respiration and intracellular adenosine triphosphate (ATP) levels were reduced whereas reactive oxygen species (ROS) production increased. Results in isolated mitochondria suggest that the mechanism responsible for these actions was a specific inhibition of complex I of the respiratory chain. The reduction in energy production triggered a compensatory mechanism mediated by the enzyme adenosine monophosphate activated protein kinase (AMPK), the master switch of cellular bio-energetics. Fluorescence and nuclear magnetic resonance demonstrated a rapid intracellular increase of neutral lipids, usually in the form of droplets. This was prevented by the AMPK inhibitor compound C and by removal of fatty acids from the culture medium. These effects were not reproduced by Nevirapine, another NNRTI. EFV is clinically coadministered with two nucleoside reverse transcriptase inhibitors. Evaluation of one of the most common combination, EFV/Lamivudine/Abacavir, revealed that the effects of EFV on ROS production were enhanced. Conclusion: Clinical concentrations of EFV induce bioenergetic stress in hepatic cells by acutely inhibiting mitochondrial function. This new mechanism of mitochondrial interference leads to an accumulation of lipids in the cytoplasm that is mediated by activation of AMPK. (HEPATOLOGY 2010;52:115-125)

Rodent models are frequently used in aging re search to investigate biochemical age effects and aid in the development of therapies for pathological and non pathological age related degenerative processes In order to validate the use of animal models in aging research and pave the way for longitudinal intervention based animal studies, the consistency of cerebral aging processes across species needs to be evaluated The dopaminergic system seems particularly susceptible to the aging process, and one of the most consistent findings in human brain aging research is a decline in striatal D2-like receptor (D2R) availability, quantifiable by positron emission tomography (PET) imaging In this study we aimed to assess whether similar age effects can be discerned in rat brains, using in vivo molecular imaging with the radioactive compound [C-11] raclopride We observed a robust decline in striatal [C-11] raclopride uptake in the aged rats in comparison to the young control group, comprising a 41% decrement in striatal binding potential In accordance with human studies, these results indicate that substantial reductions in D2R availability can be measured in the aged striatal complex Our findings suggest that rat and human brains exhibit similar biochemical alterations with age in the striatal dopaminergic system, providing support for the pertinence of rodent models in aging research (C) 2010 IBRO Published by Elsevier Ltd All rights reserved

Insulin resistance is a feature of polycystic ovary syndrome (PCOS) and is related to mitochondrial function.Our objective was to assess mitochondrial function by evaluating mitochondrial oxygen (O(2)) consumption, reactive oxygen species (ROS) production, levels of glutathione (GSH), the oxidized glutathione/GSH ratio, TNFalpha levels, and membrane potential. Additionally, we have evaluated mitochondrial complex I as a target of the oxidative stress responsible for PCOS in polymorphonuclear cells.This was a prospective controlled study conducted in an academic medical center.The study population consisted of 20 lean reproductive-age women with PCOS and 20 body composition-matched controls.We evaluated mitochondrial O(2) consumption using the Clark-type O(2) electrode; levels of ROS, GSH, and membrane potential by means of fluorescence microscopy; TNFalpha levels by ELISA; and complex I activity by spectrophotometric assay.An impairment in mitochondrial function was observed in PCOS patients, evident by a decrease in mitochondrial O(2) consumption; an increase in ROS production, oxidized glutathione/GSH ratio, and TNFalpha levels; a drop in GSH levels; and an undermining of membrane potential. Furthermore, an impairment of mitochondrial complex I was identified.This study supports the hypothesis of an association between insulin resistance and an impaired mitochondrial oxidative metabolism. We also propose that the oxidative stress responsible for PCOS takes place at complex I. These abnormalities may contribute to the increased risk of type 2 diabetes among women with PCOS.

The effects of diet supplementation with the antioxidant vitamin E (200 mg daily) on several blood neutrophil, lymphocyte and natural killer cell functions have been investigated in healthy elderly men and women before supplementation, after 3 months of supplementation and 6 months after the end of supplementation (post-supplementation). In parallel, samples of healthy adult men and women were used as age controls. In elderly men and women, an impairment of immune functions was observed in comparison with the respective adult controls and the intake of vitamin E resulted in a significant enhancement of immune parameters in both elderly men and women, bringing their values close to those in the adults. These effects were not found in post-supplementation samples in several but not in all functions. The present findings suggest that supplementation with vitamin E can produce an improvement of immune functions and therefore of health in aged people.

Antioxidants are essential, and are involved in several important biological processes such as immunity, protection against tissue damage, reproduction, growth and development. Antioxidants preserve adequate function of cells against homeostatic disturbances such as those caused by septic shock, aging and, in general, processes involving oxidative stress. Each year, many scientific articles are published describing the pharmacological and biological properties of antioxidants. This review article compiles recent findings on these properties, focusing mainly on the anti-inflammatory properties of antioxidants in different pathological areas, such as cardiovascular damage and sepsis. In relation to this process, this review focuses on the involvement of reactive oxygen and nitrogen species. Finally, the protective role of antioxidants against homeostatic disturbances such as those caused by endotoxin toxicity and cardiovascular damage, their potential clinical use, and the effects on the redox state of immune cells are discussed.

Mitochondria produce large amounts of free radicals and play an important role in the life and death of a cell. Thus, mitochondrial oxidative damage and dysfunction contribute to a number of cell pathologies that manifest themselves through a range of conditions including ischemia-reperfusion injury, sepsis, diabetes, atherosclerosis and, consequently, cardiovascular diseases (CVD). In fact, endothelial dysfunction, characterized by a loss of nitric oxide (NO) bioactivity, occurs early on in the development of atherosclerosis, and determines future vascular complications. Although the molecular mechanisms responsible for mitochondria-mediated disease processes are not yet clear, oxidative stress seems to play an important role. This review considers the process of CVD from a mitochondrial perspective. Accordingly, strategies for the targeted delivery of antioxidants to mitochondria are being developed. In this review, we will provide a summary of the following areas: the cellular metabolism of reactive oxygen species (ROS) and its role in pathophysiological processes such as CVD; currently available antioxidants and possible reasons for their efficacy and inefficacy in ameliorating oxidative stress-mediated diseases; recent developments in mitochondrially-targeted antioxidants that concentrate on the matrix-facing surface of the inner mitochondrial membrane and therefore protect against mitochondrial oxidative damage, and their therapeutic potential for future treatment of CVDs. More pre-clinical and clinical studies, however, are necessary in order to evaluate the effectiveness and toxicity of mitochondrially-targeted antioxidants.

Nitroglycerin (GTN) tolerance was induced in vivo (rats) and in vitro (rat and human vessels). Electrochemical detection revealed that the incubation dose of GTN (5×10 −6 mol/L) did not release NO or modify O 2 consumption when administered acutely. However, development of tolerance produced a decrease in both mitochondrial O 2 consumption and the K m for O 2 in animal and human vessels and endothelial cells in a noncompetitive action. GTN tolerance has been associated with impairment of GTN biotransformation through inhibition of aldehyde dehydrogenase (ALDH)-2, and with uncoupling of mitochondrial respiration. Feeding rats with mitochondrial-targeted antioxidants (mitoquinone [MQ]) and in vitro coincubation with MQ (10 −6 mol/L) or glutathione (GSH) ester (10 −4 mol/L) prevented tolerance and the effects of GTN on mitochondrial respiration and ALDH-2 activity. Biotransformation of GTN requires functionally active mitochondria and induces reactive oxygen species production and oxidative stress within this organelle, as it is inhibited by mitochondrial-targeted antioxidants and is absent in HUVECρ 0 cells. Experiments analyzing complex I–dependent respiration demonstrate that its inhibition by GTN is prevented by mitochondrial-targeted antioxidants. Furthermore, in presence of succinate (10×10 −3 mol/L), a complex II electron donor added to bypass complex I–dependent respiration, GTN-treated cells exhibited O 2 consumption rates similar to those of controls, thus suggesting that complex I was affected by GTN. We propose that, following prolonged treatment with GTN in addition to ALDH-2, complex I is a target for mitochondrially generated reactive oxygen species. Our data also suggest a role for mitochondrial-targeted antioxidants as therapeutic tools in the control of the tolerance that accompanies chronic nitrate use.

The excessive production of reactive oxygen species (ROS) associated with inflammation leads to oxidative stress, which is involved with the high mortality from several diseases such as endotoxic shock and can be controlled to a certain degree by antioxidants. The immune cells use ROS in order to support their functions and, therefore, need adequate levels of antioxidant defenses in order to avoid the harmful effect of an excessive ROS production. In the present work, the effect of the administration of the antioxidant N-acetylcysteine (NAC) on the redox state of peritoneal macrophages and lymphocytes from mice with lethal endotoxic shock (100 mg/kg i.p. of lipopolysaccharide, LPS), was studied. In both types of immune cells at 0, 2, 4, 12 and 24 h after LPS injection, an increase of ROS, of the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha), the lipid peroxidation (malonaldehyde levels, MDA), inducible nitric oxide synthase (iNOS) expression and the oxidized/reduced glutathione (GSSG/GSH) ratio, as well as a decrease of enzymatic antioxidant defenses, such as superoxide dismutase (SOD) and catalase (CAT) activity, was observed. The injection of NAC (150 mg/kg i.p. at 30 min after LPS injection) decreased the ROS, the TNFalpha the MDA levels, iNOS expression and the GSSG/GSH ratio, and increased the antioxidant defenses in both macrophages and lymphocytes. Moreover, the NAC treatment prevented the activation of nuclear translocation of the nuclear factor kappaB (NF-kappaB), which regulates ROS, inflammatory cytokines and antioxidant levels. Our present results provide evidence that both cell types have a relevant role in the pathogenesis of endotoxic shock, and that NAC, by improving the redox state of these immune cells, could increase mouse survival. Thus, antioxidants could offer an alternative treatment of human endotoxic shock.

The excessive production of reactive oxygen species (ROS), associated with inflammation, leads to a condition of oxidative stress. Oxidative stress is a major contributing factor to the high mortality rates associated with several diseases such as endotoxic shock. This condition can be controlled to a certain degree by antioxidant therapies. Immune cells use ROS in order to support their functions and therefore need adequate levels of antioxidant defenses in order to avoid the harmful effect of an excessive production of ROS. This review discusses the toxic effects of endotoxin, paying particular attention to immune function. It continues by analyzing the mechanism to which specific cells of the immune system recognize endotoxin, and the resulting pathways leading to nuclear factor-kappaB activation and proinflammatory gene transcription. We also focus on the involvement of reactive oxygen and nitric oxide (NO) and the protective role of antioxidants. The potential clinical use of antioxidants in the treatment of sepsis and the effects on the redox state of the immune cells are discussed.

In previous studies, we have observed that mice of the same strain and age show striking interindividual differences in behavior when exposed to a T-maze test. The animals that take longer to explore a T-shaped maze ("slow" animals) show high levels of emotionality/anxiety in other standard behavioral tests, prematurely aged immune functions, and a shorter life span, in comparison to "fast" mice. In these slow mice, which are a model of premature immunosenescence, the immune functions were improved after the ingestion of the thiolic antioxidant thioproline in the diet. In the present work, we studied the effects in vivo (0.1% w/w, for 4 weeks) and in vitro (0.001, 0.01, 0.1, 1, and 2.5 mM) of the thiolic antioxidant N-acetylcysteine (NAC) on different functions of peritoneal macrophages and lymphocytes from slow and fast adult Swiss mice. The results showed an improvement of all the functions studied, namely adherence to substrate, directed migration or chemotaxis, phagocytosis, and reactive oxygen species (ROS) production, after in vivo and in vitro treatment with NAC. The effect of this antioxidant was stronger in the cells from the slow than in those from the fast mice.

According to our previous work, mice of the same strain and age show striking inter-individual differences in behaviour when exposed to a T-maze test. Further, the animals exploring the maze slowly (slow mice) or staying at the starting point (freezing behaviour), which show high levels of emotionality/anxiety in other standard behavioural tests, have a less competent immune system (earlier immunosenescence) than those which explore it quickly (fast mice). The present longitudinal study on OF-1 Swiss female mice confirms and extends the above findings. Thus, the animals showing a lower performance in the T-test (slow mice) which is accompanied by a poor neuromuscular coordination in a tightrope test, have a shorter life span than the good performers (fast mice). Moreover, the slow mice have a less competent immune system as regards the following functions of peritoneal macrophages: adherence to substrate, chemotaxis, ingestion of particles and superoxide anion production. This suggests that, at the same chronological age and as regards their immune competence, the slow mice are biologically older than the fast mice. This agrees with current ideas on the close functional relationship between the nervous and the immune system in the physiological adaptation to stress, and supports the concept that an optimum level of performance of these two systems is needed to attain a long life span.

Reactive oxygen species (ROS) and proinflammatory cytokines produced by immune cells cause the oxidative stress involved in septic shock induced by endotoxin. This oxidative stress can be controlled to a certain degree by antioxidants, which is specially important for a type of immune cell, i.e. the phagocyte, that uses ROS to kill microorganisms and needs antioxidants in order to support its functions. In a previous study we have observed changes in several functions of peritoneal macrophages from BALB/c mice with lethal endotoxic shock caused by intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) (100 mg/kg), which were associated with a high production of superoxide anion. N-acetylcysteine (NAC) is a thiolic antioxidant that improves the immune response, and we have observed that when administered intraperitoneally (150 mg/kg) at 30 min after LPS injection it counteracts the effects of LPS on macrophages and lymphocytes. In the present work, we have studied the in vitro effect of several concentrations of NAC (0.001, 0.01, 0.1, 1 and 2.5 mM) on the following functions: adherence to substrate, chemotaxis, ingestion of particles, ROS production and the release of tumor necrosis factor (TNFalpha) of peritoneal macrophages from BALB/c mice at 2, 4,12 and 24 h after LPS injection. The results show that the administration of NAC (especially at 0.1 mM) decreases raised adherence, ingestion, ROS production and TNFalpha levels in macrophages from animals injected with LPS, bringing these functions to values near those of control animals. These effects which seem to be linked to a modulation of NF-kappaB, suggest that the improvement of immune functions observed in previous work after injection of NAC to animals with endotoxic shock could be due to a direct action of this thiol antioxidant on immune cells.

With aging there is an increase of oxidative stress due to an imbalance between the oxidant production and the antioxidant levels in favor of the former. Since immune cell functions are specially linked to reactive oxygen species (ROS) generation, the oxidant/antioxidant balance is essential for these cells. Although low levels of antioxidants cause a decrease in immune function, very high levels of antioxidant compounds could show prooxidant effects. In the present work, we have studied the effect of diet supplementation, for 4 weeks, with two different doses of two thiolic antioxidants, namely thioproline (TP) and N-acetylcysteine (NAC), at 0.1% (w/w) and 0.3% (w/w, of each antioxidant) on the main immune system cells, i.e.: macrophages, lymphocytes and natural killer (NK) cells of adult (33+/-1 week old) and aged (75+/-1 week old) female Swiss mice. Two groups of animals, adult and aged mice, fed standard diet were used as controls. The results show that the ingestion of 0.1% doses of thiols improves, in the adult mice, several immune functions such as the chemotaxis capacity of both macrophages and lymphocytes, the phagocytosis of macrophages, the lymphoproliferative response to the mitogen Con A and the NK activity. Moreover, no change was observed in adherence capacity of immune cells, and superoxide production was decreased. By contrast, in aged mice the ingestion of these amounts of antioxidants did not change the immune functions studied with the exception of NK activity, which was stimulated. The ingestion of 0.3% of antioxidants by adult mice only increased some immune functions such as adherence and superoxide production, which are markers of oxidative stress. Other functions such as chemotaxis or lymphoproliferative response decreased. However, the ingestion of these very high amounts of thiols by aged animals increased the phagocytosis, the NK activity and specially the lymphoproliferative response to the mitogen, a function that is very depressed with aging.

Aging associates with a decline of physiological functions, including the function of the nervous and the immune system. These aged-related changes occur in various degrees in different members of a mouse outbred population. Accordingly, we have proposed a model of premature immunosenescence in mice, based on the demonstration of premature decline in the behavioral response in a simple T-maze and in several immune functions in Swiss outbred mice. Those mice with a worst (slow) performance in this test (linked to a higher emotional response to stress) show a shorter life span and a decreased immune function when compared to fast mice. In order to provide biomarkers of "biological aging" related to health and survival, the present longitudinal study includes the analysis of several immunological parameters such as, proliferative response to mitogen Con A, NK activity and cytokine (TNFalpha, IL-1beta and IL-2) release by peritoneal leukocytes from female Swiss mice. Slow mice showed a lower proliferative response to Con A, IL-2 and IL-1beta release, an impaired NK activity and an increased TNFalpha production as compared to fast mice. Moreover, the age-associated decline of these functions is more strikingly slow than in fast mice. In summary, we propose the above immunological parameters, that change with aging at a different rate in members of a same population, as useful biomarkers to asses the rate of biological aging in mice.

ContextAnorexia nervosa is a common illness among adolescents and is characterised by oxidative stress.ObjectiveThe effects of anorexia on mitochondrial function and redox state in leukocytes from anorexic subjects were evaluated.Design and settingA multi-centre, cross-sectional case-control study was performed.PatientsOur study population consisted of 20 anorexic patients and 20 age-matched controls, all of which were Caucasian women.Main outcome measuresAnthropometric and metabolic parameters were evaluated in the study population. To assess whether anorexia nervosa affects mitochondrial function and redox state in leukocytes of anorexic patients, we measured mitochondrial oxygen consumption, membrane potential, reactive oxygen species production, glutathione levels, mitochondrial mass, and complex I and III activity in polymorphonuclear cells.ResultsMitochondrial function was impaired in the leukocytes of the anorexic patients. This was evident in a decrease in mitochondrial O2 consumption (PConclusionsOxidative stress is produced in the leukocytes of anorexic patients and is closely related to mitochondrial dysfunction. Our results lead us to propose that the oxidative stress that occurs in anorexia takes place at mitochondrial complex I. Future research concerning mitochondrial dysfunction and oxidative stress should aim to determine the physiological mechanism involved in this effect and the physiological impact of anorexia.

Oxidative stress associated with reactive oxygen species (ROS) and cytokines produced by immune cells, which is involved in septic shock caused by endotoxin, can be controlled to a certain degree by antioxidants with free radical scavenging action. N-acetylcysteine (NAC) and ascorbic acid (AA) are ROS scavengers that improve the immune response, and modulate macrophage function in mice with endotoxin-caused oxidative stress. Therefore, we have investigated the in vitro effects of these antioxidants on the functions of lymphocytes from BALB/c mice with lethal endotoxic shock caused by intraperitoneal injection of E. coli lipopolysaccharide (LPS) (100 mg/kg). Adherence to tissues and chemotaxis (the earliest two functions of lymphocytes in the immune response), as well as ROS levels and TNF alpha production were determined in the presence or absence of NAC or AA (0.001, 0.01, 0.1, 1 and 2.5 mM) in lymphocytes from peritoneum, axillary nodes, spleen and thymus obtained at several times (2, 4, 12 and 24 hours) after LPS injection. Endotoxic shock decreases the chemotaxis of lymphocytes from all the above localizations and increases their adherence, TNF alpha and ROS production. These changes in lymphocyte function were counteracted by NAC and AA, bringing these functions to values near those of control animals. Our data suggest that lymphocytes are important targets of endotoxins contributing to oxidative stress by septic shock, and that antioxidants can preserve the function of lymphocytes, preventing the homeostatic disturbances caused by endotoxin.

Changes in the bidirectional interaction between the nervous and the immune systems have been proposed as a cause of ageing. Neuropeptides, such as neuropeptide Y (NPY), could show different effects on immune function with age. In the present work, we have studied the in vitro action of a wide range of NPY concentrations, i.e. from 10(-13) to 10(-7) M, on natural killer (NK) activity, a function which decreases with age. Spleen, axillary nodes, thymus and peritoneum leukocytes from mice of different ages: young (12+/-2 weeks), adult (24+/-2 weeks), mature (50+/-2 weeks) and old (72+/-2 weeks) were used. Stimulation by NPY of NK activity was observed in adult and mature animals in axillary nodes and thymus, and an inhibition in the spleen from young mice. The specificity of the NPY effect on cytotoxic activity was confirmed using a C-terminal fragment of NPY. Furthermore, cAMP levels in leukocytes were found to be decreased by NPY in adult mice, suggesting an involvement of this messenger system in the NK modulation by this neuropeptide.

Retinol binding protein 4 (RBP4) is an adipokine that may contribute to the development of insulin resistance. However, how this adipokine is affected and its possible involvement in lipid metabolism in obese patients with varying degrees of insulin resistance is yet to be determined. A total of 299 middle-aged morbid obese patients (BMI>40 kg/m(2)) were divided in euglycemic, metabolic syndrome or type 2 diabetic. Anthropometric measurements, biochemical variables and systemic RBP4 levels were determined. RBP4 levels were significantly higher in patients with metabolic syndrome and type 2 diabetes than in euglycemic subjects (42.9±14.6; 42.3±17.0 and 37.4±11.7 µg/ml, respectively) and correlated with triglycerides but not with those of HOMA-IR in the whole population. The multivariate regression model revealed that triglycerides were the strongest predictor of systemic RBP4 levels. Analysis of lipoprotein subfractions in a subpopulation of 80 subjects showed an altered profile of insulin resistant states characterized by higher VLDL, sdLDL and small HDL percentages and lower large HDL percentage. Although RBP4 levels correlated significantly with LDL particle size and small HDL percentage, the latter parameter was independently associated only with RBP4. Our study reveals that systemic RBP4 levels could play an important role in lipid metabolism in morbid obesity, increasing triglyceride levels and contributing to the formation of small HDL.

Summary of results EFV produced an immediate reduction of mitochondrialfunction, evident by the significant and dose-dependentinhibition of mitochondrial O2 consumption and thedecrease of intracellular ATP and Δψm. This metabolicstress promoted the activation of AMPK, triggering severalof its signalling pathways, as EFV induced an increment inCD36 mRNA expression and in intracellular lipid content,which could have been a result of the formation of lipiddroplets. This intracellular lipid increase was not presentin cells treated with Compound C, which points to a keyrole for AMPK in these mechanisms. Conclusion Given that EFV treatment is usually prolonged, thesemechanisms may effect the general regulation of lipidmetabolism and could cause the alterations that are char-acteristic of lipoatrophy. References 1. Blanco F: Involvement of Efavirenz in Lipodystrophy. AIDSReviews 2004, 6: 117-9.2. Viollet B, et al. : Activation of AMP-activated protein kinase inthe liver: a new strategy for the management of metabolichepatic disorders.