Anabel García-Heredia

Xenohormetic and anti-aging activity of secoiridoid polyphenols present in extra virgin olive oil: a new family of gerosuppressant agents.

Aging can be viewed as a quasi-programmed phenomenon driven by the overactivation of the nutrient-sensing mTOR gerogene. mTOR-driven aging can be triggered or accelerated by a decline or loss of responsiveness to activation of the energy-sensing protein AMPK, a critical gerosuppressor of mTOR. The occurrence of age-related diseases, therefore, reflects the synergistic interaction between our evolutionary path to sedentarism, which chronically increases a number of mTOR activating gero-promoters (e.g., food, growth factors, cytokines and insulin) and the “defective design” of central metabolic integrators such as mTOR and AMPK. Our laboratories at the Bioactive Food Component Platform in Spain have initiated a systematic approach to molecularly elucidate and clinically explore whether the “xenohormesis hypothesis,” which states that stress-induced synthesis of plant polyphenols and many other phytochemicals provides an environmental chemical signature that upregulates stress-resistance pathways in plant consumers, can be explained in terms of the reactivity of the AMPK/mTOR-axis to so-called xenohormetins. Here, we explore the AMPK/mTOR-xenohormetic nature of complex polyphenols naturally present in extra virgin olive oil (EVOO), a pivotal component of the Mediterranean style diet that has been repeatedly associated with a reduction in age-related morbid conditions and longer life expectancy. Using crude EVOO phenolic extracts highly enriched in the secoiridoids oleuropein aglycon and decarboxymethyl oleuropein aglycon, we show for the first time that (1) the anticancer activity of EVOO secoiridoids is related to the activation of anti-aging/cellular stress-like gene signatures, including endoplasmic reticulum (ER) stress and the unfolded protein response, spermidine and polyamine metabolism, sirtuin-1 (SIRT1) and NRF2 signaling; (2) EVOO secoiridoids activate AMPK and suppress crucial genes involved in the Warburg effect and the self-renewal capacity of “immortal” cancer stem cells; (3) EVOO secoiridoids prevent age-related changes in the cell size, morphological heterogeneity, arrayed cell arrangement and senescence-associated β-galactosidase staining of normal diploid human fibroblasts at the end of their proliferative lifespans. EVOO secoiridoids, which provide an effective defense against plant attack by herbivores and pathogens, are bona fide xenohormetins that are able to activate the gerosuppressor AMPK and trigger numerous resveratrol-like anti-aging transcriptomic signatures. As such, EVOO secoiridoids constitute a new family of plant-produced gerosuppressant agents that molecularly “repair” the aimless (and harmful) AMPK/mTOR-driven quasi-program that leads to aging and aging-related diseases, including cancer.

Impaired paraoxonase-1 status in obese children. Relationships with insulin resistance and metabolic syndrome

OBJECTIVES To investigate the relationships between serum paraoxonase-1 (PON1), insulin resistance, and metabolic syndrome (MetS) in childhood obesity. DESIGN AND METHODS We studied 110 obese children and 36 non-obese children with a similar gender and age distribution. We measured serum PON1 activity against 5-thiobutyl butyrolactone (TBBLase) and against paraoxon (paraoxonase). PON1 concentration was measured separately as were the levels of several standard metabolic variables. The homeostasis model assessment (HOMA) index was calculated as an estimate of insulin resistance. RESULTS TBBLase was significantly decreased in obese children (P=0.008), while paraoxonase activity and PON1 concentrations showed non-significant trends towards decrease and increase, respectively (P=0.054 and P=0.060). TBBLase and paraoxonase specific activities were significantly decreased (P=0.004 and P=0.018, respectively). TBBLase specific activity was inversely associated with BMI, percentage body fat, insulin, HOMA, triglycerides, and C-reactive protein, and directly associated with HDL-cholesterol. Paraoxonase specific activity showed similar associations with BMI, percentage fat, HDL-cholesterol, and C-reactive protein. Obese children with MetS had lower TBBLase activities than obese children without MetS (P=0.018). Linear regression analyses showed that TBBLase was independently associated with HDL-cholesterol, BMI, percentage body fat and PON155 polymorphism, but paraoxonase activity was associated only with PON1192 polymorphism. CONCLUSIONS Our results suggest that PON1 may play a role in the onset and development of metabolic alterations in childhood obesity leading to diabetes and cardiovascular disease later in life. However, being derived from statistical association study, this finding cannot be seen as showing cause-effect.

Paraoxonase-1 Deficiency Is Associated with Severe Liver Steatosis in Mice Fed a High-fat High-cholesterol Diet: A Metabolomic Approach

Oxidative stress is a determinant of liver steatosis and the progression to more severe forms of disease. The present study investigated the effect of paraoxonase-1 (PON1) deficiency on histological alterations and hepatic metabolism in mice fed a high-fat high-cholesterol diet. We performed nontargeted metabolomics on liver tissues from 8 male PON1-deficient mice and 8 wild-type animals fed a high-fat, high-cholesterol diet for 22 weeks. We also measured 8-oxo-20-deoxyguanosine, reduced and oxidized glutathione, malondialdehyde, 8-isoprostanes and protein carbonyl concentrations. Results indicated lipid droplets in 14.5% of the hepatocytes of wild-type mice and in 83.3% of the PON1-deficient animals (P < 0.001). The metabolomic assay included 322 biochemical compounds, 169 of which were significantly decreased and 16 increased in PON1-deficient mice. There were significant increases in lipid peroxide concentrations and oxidative stress markers. We also found decreased glycolysis and the Krebs cycle. The urea cycle was decreased, and the pyrimidine cycle had a significant increase in orotate. The pathways of triglyceride and phospholipid synthesis were significantly increased. We conclude that PON1 deficiency is associated with oxidative stress and metabolic alterations leading to steatosis in the livers of mice receiving a high-fat high-cholesterol diet.

Rosiglitazone and fenofibrate exacerbate liver steatosis in a mouse model of obesity and hyperlipidemia. A transcriptomic and metabolomic study.

Peroxisome proliferator-activated receptors (PPAR) play an important role in the regulation of lipid and glucose metabolism, inflammatory, and vascular responses. We show the effect of treatment with two PPAR agonists, fenofibrate (FF) and rosiglitazone (RSG), on ob/ob and LDLR-double deficient mice, by combined gene-expression and metabolomic analyses. Male mice were daily treated for 12 weeks with RSG (10 mg·kg(1-)·day(-1) per os (p.o.), n = 8) and FF (50 mg·kg(1-)·day(-1) p.o., n = 8). Twelve untreated ob/ob and LDLR-double deficient mice were used as controls. To integrate the transcriptomic and metabolomic results, we designed a hierarchical algorithm, based on the average linkage method in clustering. Data were also interpreted with the Ingenuity Pathway Analysis program. FF and RSG treatments significantly increased the hepatic triglyceride content in the liver when compared with the control group, and the treatments induced an increase in the number and size of hepatic lipid droplets. Both drugs simultaneously activate pro-steatotic and antisteatotic metabolic pathways with a well-ordered result of aggravation of the hepatic lipid accumulation. The present study is a cautionary note not only to researchers on the basic mechanism of the action of PPAR activators but also to the use of these compounds in clinical practice.

PPARs in Regulation of Paraoxonases: Control of Oxidative Stress and Inflammation Pathways

The paraoxonase (PON) group of enzymes, composed of PON1, PON2, and PON3, play an important role in decreasing oxidative stress by degrading lipid peroxides. PON1 synthesis is upregulated by PPAR. Several pharmacological compounds (acting as antioxidants and, hence, atheroprotective) stimulate both PPAR activity and PON1 expression. Recent evidence suggests that PON1 and the monocyte chemoattractant protein-1 (MCP-1) are involved in coordinating the inflammatory response in damaged tissues; PPAR may be central in the regulation of these biochemical pathways. This article reviews the state of knowledge on PON1 biochemistry and function, the influence of genetic variation, and the regulation of PON1 expression by pharmaceutical compounds that increase PPAR activity. We also describe recent lines of evidence suggesting links between PON1 and MCP-1 and how their production may be regulated by PPAR.

Introduction: Oxidation and Inflammation, A Molecular Link Between Non-communicable Diseases

Non-communicable diseases are, by definition, those chronic diseases that are non-infectious and non-transmissible. The most common non-communicable diseases are obesity, diabetes, cancer, and cardiovascular, chronic respiratory and neurological diseases. Altogether, they are the commonest cause of death and disability in modern world. Recent investigations show that many of these diseases share common pathophysiological mechanisms and are, at least in part, different manifestations in different organs of similar molecular alterations. Mitochondrial alterations, oxidative stress and inflammation are inextricably linked and play major roles in the onset and development of non-communicable diseases. Therefore, it is conceivable that pharmacological or nutritional manipulation of oxidation and inflammation allows a significant decrease in the mortality and morbility associated to these diseases.

Proteomic analysis reveals oxidative stress response as the main adaptative physiological mechanism in cows under different production systems.

Three groups of cows representing three ranges of welfare in the production system were included in the study: two groups of Bruna dels Pirineus beef cattle maintained under different management systems (good and semiferal conditions) and a group of Alberes cows, a breed that lives in the mountains (hardest conditions). In order to identify new stress/welfare biomarkers, serum from Bruna cows living in both environments was subjected to DIGE labelling, two-dimensional electrophoresis and MALDI-MS or ion trap MS. Identification was achieved for 15 proteins, which mainly belonged to three biological functions, the oxidative stress pathway (glutathione peroxidase (GPx) and paraoxonase (PON-1)), the acute phase protein family (Heremans Schmid glycoprotein alpha2 (α2-HSG)) and the complement system. Biological validation included the Alberes breed. GPx and PON-1 were validated by an enzymatic assay and found to be higher and lower, respectively, in cows living in hard conditions. α2-HSG was validated by ELISA and found to be reduced in hard conditions. Other biomarkers of the redox status were also altered by living conditions: protein carbonyl content, superoxide dismutase (SOD) and glutathione reductase (GR). Our results show that changes in the redox system are the main adaptation of cows living in challenging environmental conditions.

Paraoxonases and Chemokine (C–C Motif) Ligand-2 in Noncommunicable Diseases

Oxidative stress and inflammation underpin most diseases; their mechanisms are inextricably linked. Chronic inflammation is associated with oxidation, anti-inflammatory cascades are linked to decreased oxidation, increased oxidative stress triggers inflammation, and redox balance inhibits the inflammatory cellular response. Whether or not oxidative stress and inflammation represent the cause or consequence of cellular pathology, they contribute significantly to the pathogenesis of noncommunicable diseases (NCD). The incidence of obesity and other related metabolic disturbances are increasing, as are age-related diseases due to a progressively aging population. Relationships between oxidative stress, inflammatory signaling, and metabolism are, in the broad sense of energy transformation, being increasingly recognized as part of the problem in NCD. In this chapter, we summarize the pathologic consequences of an imbalance between circulating and cellular paraoxonases, the system for scavenging excessive reactive oxygen species and circulating chemokines. They act as inducers of migration and infiltration of immune cells in target tissues as well as in the pathogenesis of disease that perturbs normal metabolic function. This disruption involves pathways controlling lipid and glucose homeostasis as well as metabolically driven chronic inflammatory states that encompass several response pathways. Dysfunction in the endoplasmic reticulum and/or mitochondria represents an important feature of chronic disease linked to oxidation and inflammation seen as self-reinforcing in NCD. Therefore, correct management requires a thorough understanding of these relationships and precise interpretation of laboratory test results.

Paraoxonase-1 Inhibits Oxidized Low-Density Lipoprotein-Induced Metabolic Alterations and Apoptosis in Endothelial Cells: A Nondirected Metabolomic Study

We studied the influence of PON1 on metabolic alterations induced by oxidized LDL when incubated with endothelial cells. HUVEC cells were incubated with native LDL, oxidized LDL, oxidized LDL plus HDL from wild type mice, and oxidized LDL plus HDL from PON1-deficient mice. Results showed alterations in carbohydrate and phospholipid metabolism and increased apoptosis in cells incubated with oxidized LDL. These changes were partially prevented by wild type mouse HDL, but the effects were less effective with HDL from PON1-deficient mice. Our results suggest that PON1 may play a significant role in endothelial cell survival by protecting cells from alterations in the respiratory chain induced by oxidized LDL. These results extend current knowledge on the protective role of HDL and PON1 against oxidation and apoptosis in endothelial cells.

Paraoxonase-1 status in patients with hereditary hemochromatosis

Hereditary hemochromatosis (HH) is characterized by accumulation of iron, oxidative stress, inflammation, and fibrogenesis in liver tissue. In this setting, research on the protection afforded by intracellular antioxidants is of clinical relevance. Paraoxonase-1 (PON1) is an enzyme that degrades lipid peroxides. This study investigates the alterations in serum PON1 status, PON1 gene polymorphisms, and PON1 hepatic expression in patients with HH. We performed a case-control study in 77 patients with HH (80.5% men, 22–70 years of age) and 408 healthy individuals (43.1% men, 26–74 years of age). Serum PON1 activities against different substrates and PON1192 and PON155 polymorphisms were analyzed. PON1 protein expression was investigated in 20 liver biopsies. HH patients had significantly lower serum PON1 activity, which was inversely correlated with ferritin (marker of iron stores) and serum 8-isoprostane concentrations (index of oxidative stress). PON1 protein expression in liver tissue was higher in patients and showed stronger staining in hepatocytes surrounding the areas of inflammation. Our study provides preliminary evidence that PON1 may play a role in protecting against iron-induced oxidative stress in hereditary hemochromatosis.