Paraoxonase 2 Research Articles

Paraoxonase-2 variants potentially influence insulin resistance, beta-cell function, and their interrelationships with alanine aminotransferase in type 2 diabetes.

Background:The aim of this study was to determine whether insulin resistance, beta-cell function, and their associations with alanine aminotransferase (ALT) are affected by the functional variants of paraoxonase-2 (PON2) as an intracellular antioxidant in patients with type 2 diabetes (T2D).Materials and Methods:Quantitative insulin sensitivity check index (QUICKI) and homeostasis model assessment for beta-cell function (HOMA-BCF) were assessed in T2D patients. Insulin levels were determined using ELISA. The variants PON2-A148G and PON2-S311C were genotyped using polymerase chain reaction-based restriction fragment length polymorphism.Results:According to the PON2-G148A variant, ALT was found to be significantly correlated with QUICKI (r = −0.616, P = 0.005) and HOMA-BCF (r = 0.573, P = 0.01) in the GA + GG group; however, the correlations were not statistically significant in the AA genotypes. Based on the genotypes of PON2-S311C, there was a significant correlation between ALT with QUICKI (r = −0.540, P = 0.031) and HOMA-BCF (r = 0.567, P = 0.022) in the SC + CC group. In the multiple adjusted logistic regression analyses, considering the variants PON2-G148A and PON2-C311S as independent variables and QUICKI and HOMA-BCF as the dependent variables, both variants were significantly associated with the QUICKI (P = 0.019 for PON2-G148A and P = 0.041 for PON2-C311S). Furthermore, PON2-C311S remained significantly associated with HOMA-BCF (P = 0.03).Conclusion:These data implicate a role for the functional variants of PON2 in insulin resistance and beta-cell function as well as underscore the effective role of these variants in the associations between them and ALT. Our data contribute to our understanding of the important physiologic functions of PON2 in glucose metabolism and its related metabolic diseases.

PON2 Exemplifies a Unique Dependency of B Cell Lineage ALL Cells on Detoxifying Lactonases

Background and significance: Paraoxonase 2 (PON2) is a member of mammalian detoxifying enzymes that are located to the mitochondrial membrane, hydrolyze lactone metabolites and interact with coenzyme Q10 to diminish oxidative stress. While PON2 is highly expressed in the CNS and multiple fetal tissues, expression levels in normal hematopoietic cells are low. We began to study the function of PON2 in B cell lineage ALL, because microarray analyses suggested high mRNA levels of PON2 in B cell lineage ALL cells. In addition, PON2 is used as a diagnostic marker on a 15 gene diagnostic LDA panel (e.g. in AALL1131; NCT02883049) that is used for the identification of Ph-like ALL, a subgroup with particularly poor outcome and specific treatment requirements.Results: Analyzing data from pediatric and adult clinical trials, we found that greater than median PON2 mRNA levels at the time of diagnosis predicted poor clinical outcomes for both children (COG P9906; n=207; P=1.09e-05) and adults (ECOG; n=215; P=0.003) with B-lineage ALL. Studying expression levels of PON2 by quantitative RT-PCR and Western blot in normal bone marrow B cell precursors from healthy donors and patient-derived pre-B ALL cells including Ph+ and Ph -like ALL, we found 3-10-fold increased mRNA and protein levels of PON2 throughout multiple pre-B ALL samples. To elucidate potential functions of PON2 in normal B cells and B-lineage ALL, we studied normal B cell development and ALL-models in Pon2-/- mice. While B cell development was unperturbed in Pon2 -deficient mice, deletion of Pon2 had profound effects on both BCR-ABL1- and N RASG12D-driven leukemogenesis. Compared to wildtype, Pon2-/- pre-B ALL cells failed to form colonies in semisolid agar. Pon2-/-ALL cells were arrested in G0/G1 phase and expressed 2-5-fold increased levels of Arf and p21, compared to Pon2+/+ ALL cells. Strikingly, >50% of Pon2-/-ALL cells spontaneously underwent cellular senescence, as shown by b-galactosidase staining in conjunction with increased Arf expression levels. These in vitro findings suggest an important role of PON2 in B-lymphoid leukemogenesis, which was confirmed in transplant experiments based on BCR-ABL1 and N RASG12D ALL models. While Pon2 -deficiency substantially prolonged survival of recipient mice of BCR-ABL1 ALL cells (P=0.0001), mice transplanted with Pon2 -deficient N RASG12D ALL cells survived for indefinite periods of time.Targeting PON2 expression in relapse ALL: Given that PON2 is expressed at very high levels in relapse ALL samples, we tested the concept of targeting PON2 lactonase activity in a prodrug-approach. While PON2 activity typically results in detoxification of lactone-metabolites, lactone-hydrolysis of the N-(3-oxododecanoyl)-homoserine lactone (3OC12) prodrug results in cytotoxic byproducts (Guoping et al., 2016). We therefore tested the therapeutic potential of 3OC12. For genetic validation, we treated wildtype and Pon2-/- ALL cells with 3OC12 and found strong cytotoxic effects in wildtype but not Pon2-/- ALL cells. Likewise, inducible overexpression of Pon2 in patient-derived pre-B ALL cells exacerbated toxicity of 3OC12 compared to empty vector controls. Likewise, CRISPR-Cas9 mediated ablation of PON2 in human ALL PDX reversed sensitivity of the pre-B ALL cells to 3OC12.Conclusion: Here we describe the previously unknown function of the detoxifying PON2 lactonase as an essential prerequisite for pre-B cell transformation and leukemogenesis. PON2 expression is specific for leukemia cells, an outcome predictor for patients with pre-B ALL and a biomarker of Ph-like ALL. While PON2 protects ALL cells and enables malignant growth, we demonstrate that its lactonase-activity can be leveraged for pharmacological targeting as exemplified by the lactone-prodrug 3OC12. DisclosuresNo relevant conflicts of interest to declare.

Effect of certain non-steroidal anti-inflammatory drugs on the paraoxonase 2 (PON2) in human monocytic cell line U937

The paraoxonase gene family in humans consists of three members as PON1, PON2 and PON3. PON2 can be expressed in several tissues; however, it is not released from the cells in those tissues. PON2 is also expressed in macrophages. Firstly, the commonly used NSAIDs diclofenac sodium and tenoxicam were applied on U937 cell line, the in vitro human monocyte cell line. Than PON2 specific Lactonase activity and paraoxonase family specific arylesterase were determined. Use of Diclofenac sodium in 0.845 mM dose during 6–12 h of incubation and Tenoxicam in 0.74 mM dose during 6 h of incubation resulted in a significant decline in the lactonase activity. Diclofenac sodium didn’t make any change in the arylesterase activity. On the other hand, tenoxicam decreased arylesterase activity during the use of 12 h, in 0.74 mM and 1.48 mM dose.

Regulation of the epithelial Na+ channel by paraoxonase-2

Paraoxonase-2 (PON-2) is a membrane-bound lactonase with unique anti-oxidative and anti-atherosclerotic properties. PON-2 shares key structural elements with MEC-6, an endoplasmic reticulum-resident molecular chaperone in Caenorhabditis elegans MEC-6 modulates the expression of a mechanotransductive ion channel comprising MEC-4 and MEC-10 in touch-receptor neurons. Because pon-2 mRNA resides in multiple rat nephron segments, including the aldosterone-sensitive distal nephron where the epithelial Na+ channel (ENaC) is expressed, we hypothesized that PON-2 would similarly regulate ENaC expression. We observed PON-2 expression in aquaporin 2-positive principal cells of the distal nephron of adult human kidney. PON-2 also co-immunoprecipitated with ENaC when co-expressed in HEK293 cells. When PON-2 was co-expressed with ENaC in Xenopus oocytes, ENaC activity was reduced, reflecting a reduction in ENaC surface expression. MEC-6 also reduced ENaC activity when co-expressed in Xenopus oocytes. The PON-2 inhibitory effect was ENaC-specific, as PON-2 had no effect on functional expression of the renal outer medullary potassium channel. PON-2 did not alter the response of ENaC to extracellular Na+, mechanical shear stress, or α-chymotrypsin-mediated proteolysis, suggesting that PON-2 did not alter the regulation of ENaC by these factors. Together, our data suggest that PON-2 regulates ENaC activity by modulating its intracellular trafficking and surface expression.

Paraoxonase 2 Facilitates Pancreatic Cancer Growth and Metastasis by Stimulating GLUT1-Mediated Glucose Transport

Metabolic deregulation is a hallmark of human cancers, and the glycolytic and glutamine metabolism pathways were shown to be deregulated in pancreatic ductal adenocarcinoma (PDAC). To identify new metabolic regulators of PDAC tumor growth and metastasis, we systematically knocked down metabolic genes that were overexpressed inhuman PDAC tumor samples using short hairpin RNAs. We found that p53 transcriptionally represses paraoxonase 2 (PON2), which regulates GLUT1-mediated glucose transport via stomatin. The loss of PON2 initiates the cellular starvation response and activates AMP-activated protein kinase (AMPK). In turn, AMPK activates FOXO3A and its transcriptional target, PUMA, which inducesanoikis to suppress PDAC tumor growth and metastasis. Pharmacological or genetic activation of AMPK, similar to PON2 inhibition, blocks PDAC tumor growth. Collectively, our results identify PON2 as a new modulator of glucose transportthat regulates a pharmacologically tractable pathway necessary for PDAC tumor growth and metastasis.

Decreased paraoxonase 2 enzymatic activity in monocyte/macrophages cells. A comparative in vivo and in vitro study for diabetes

Aim: To investigate peripheral blood monocytes/macrophages (Mo/Mᴓ) paraoxonase 2 (PON2) in diabetes and the factors modulating its activity.Methods: One hundred and eighteen patients with newly diagnosed uncomplicated type 2 diabetes mellitus were compared regarding clinical, biochemical and oxidative stress parameters with 80 healthy subjects. The capacity of the peripheral blood mononuclear cells (PBMNC) to release pro-oxidants and to neutralise them was determined by measuring the respiratory burst (RB) and the intracellular antioxidant enzyme PON2. In vitro experiments were conducted on a differentiated monocytes cell line (dU937) that was exposed to serum deprivation followed by addition of isolated lipoproteins (VLDL or LDL).Results: Paraoxonase 2 activity in Mo/Mᴓ was significantly lower in type 2 diabetes patients (0.042 ± 0.044 vs 0.165 ± 0.133U lactonase activity/mg protein in controls, p < .0005) and decreased in the obese in all groups. It was inversely correlated to parameters of adiposity (BMI and Waist Circumference), of glucose control (blood glucose, fructosamine and HbA1c) and insulin resistance (HOMA-IR). In multivariate regression models, 15–34% of the PON2 variance was explained by diabetes. The in vitro addition of VLDL normalised the RB of serum deprived dU937 cells, S− (to 82 ± 18% of the cells incubated with serum, S+) and PON2 activity (from 0.524 ± 0.061 in S − to 0.298 ± 0.048 U/mg protein). In contrast, when LDL was added, the RB remained lower (61 ± 12% of S+, p = .03) and PON2 higher (0.580 ± 0.030 U/mg protein, p = .003).Conclusions: The decrease in monocyte/macrophage PON2 enzymatic activity observed in type 2 diabetes cannot be totally explained by abdominal obesity and insulin resistance. The underlying molecular mechanisms need to be identified.

Exploring the role of Paraoxonase-2 in bladder cancer: analyses performed on tissue samples, urines and cell culturess

The enzyme paraoxonase-2 (PON2) is ubiquitously expressed and exerts its antiapoptotic and antioxidative functions in several intracellular compartments.The aim of this study is to investigate the role of PON2 in bladder cancer (BC). The expression levels of PON2 in paired tumor and normal bladder tissue samples and in urinary exfoliated cells from patients affected with BC and healthy donors were evaluated. Moreover, the effect of PON2 overexpression on tumor cell proliferation and susceptibility to oxidative stress was investigated in human bladder cancer cell line T24.Our results showed that PON2 expression levels were significantly higher in BC compared with non-tumor tissue. In urinary exfoliated cells from BC patients, PON2 mRNA levels showed an inverse correlation with tumor stage (pT). Moreover, PON2 overexpression in T24 cells led to a significant increase in tumor cell proliferation and resistance to oxidative stress.The results obtained showed that PON2 could represent a molecular biomarker for bladder cancer and suggest a potential role of the enzyme as a prognostic factor for this neoplasm.

Paraoxonase: The Universal Factor of Antioxidant Defense in Human Body

The paraoxonase (PON) gene family includes three members: PON1, PON2, and PON3 aligned in tandem on chromosome 7 in humans. All PON proteins share considerable structural homology and have the capacity to protect cells from oxidative stress; therefore, they have been implicated in the pathogenesis of several inflammatory diseases, particularly atherosclerosis. Increased production of reactive oxygen species as a result of decreased activities of mitochondrial electron transport chain complexes plays a role in the development of many inflammatory diseases, including atherosclerosis. PON1 and PON3 proteins can be detected in plasma and reside in the high-density lipoprotein fraction and protect against oxidative stress by hydrolyzing certain oxidized lipids in lipoproteins, macrophages, and atherosclerotic lesions. Paraoxonase 2 (PON2) possesses antiatherogenic properties and is associated with lower ROS levels. PON2 is involved in the antioxidative and anti-inflammatory response in intestinal epithelial cells. In contrast to PON1 and PON3, PON2 is cell-associated and is not found in plasma. It is widely expressed in a variety of tissues, including the kidney, and protects against cellular oxidative stress. Overexpression of PON2 reduces oxidative status, prevents apoptosis in vascular endothelial cells, and inhibits cell-mediated low density lipoprotein oxidation. PON2 also inhibits the development of atherosclerosis, via mechanisms involving the reduction of oxidative stress. In this review we explore the physiological roles of PON in disease development and modulation of PONs by infective (bacterial, viral) agents.

Valproic acid inhibits glioblastoma multiforme cell growth via paraoxonase 2 expression.

We studied the potential mechanisms of valproic acid (VPA) in the treatment of glioblastoma multiforme (GBM). Using the human U87, GBM8401, and DBTRG-05MG GBM-derived cell lines, VPA at concentrations of 5 to 20 mM induced G2/M cell cycle arrest and increased the production of reactive oxygen species (ROS). Stress-related molecules such as paraoxonase 2 (PON2), cyclin B1, cdc2, and Bcl-xL were downregulated, but p27, p21 and Bim were upregulated by VPA treatment. VPA response element on the PON2 promoter was localized at position -400/−1. PON2 protein expression was increased in GBM cells compared with normal brain tissue and there was a negative correlation between the expression of PON2 and Bim. These findings were confirmed by the public Bredel GBM microarray (Gene Expression Omnibus accession: GSE2223) and the Cancer Genome Atlas GBM microarray datasets. Overexpression of PON2 in GBM cells significantly decreased intracellular ROS levels, and PON2 expression was decreased after VPA stimulation compared with controls. Bim expression was significantly induced by VPA in GBM cells with PON2 silencing. These observations were further shown in the subcutaneous GBM8401 cell xenograft of BALB/c nude mice. Our results suggest that VPA reduces PON2 expression in GBM cells, which in turn increases ROS production and induces Bim production that inhibits cancer progression via the PON2–Bim cascade.

Sex and genetic differences in the effects of acute diesel exhaust exposure on inflammation and oxidative stress in mouse brain.

In addition to increased morbidity and mortality caused by respiratory and cardiovascular diseases, air pollution may also contribute to central nervous system (CNS) diseases. Traffic-related air pollution is a major contributor to global air pollution, and diesel exhaust (DE) is its most important component. DE contains more than 40 toxic air pollutants and is a major constituent of ambient particulate matter (PM), particularly of ultrafine-PM. Limited information suggests that exposure to DE may cause oxidative stress and neuroinflammation in the CNS. We hypothesized that males may be more susceptible than females to DE neurotoxicity, because of a lower level of expression of paraoxonase 2 (PON2), an intracellular anti-oxidant and anti-inflammatory enzyme. Acute exposure of C57BL/6 mice to DE (250-300μg/m3 for 6h) caused significant increases in lipid peroxidation and of pro-inflammatory cytokines (IL-1α, IL-1β, IL-3, IL-6, TNF-α) in various brain regions (particularly olfactory bulb and hippocampus). In a number of cases the observed effects were more pronounced in male than in female mice. DE exposure also caused microglia activation, as measured by increased Iba1 (ionized calcium-binding adapter molecule 1) expression, and of TSPO (translocator protein) binding. Mice heterozygotes for the modifier subunit of glutamate cysteine ligase (the limiting enzyme in glutathione biosynthesis; Gclm+/- mice) appeared to be significantly more susceptible to DE-induced neuroinflammation than wild type mice. These findings indicate that acute exposure to DE causes neuroinflammation and oxidative stress in brain, and suggest that sex and genetic background may play important roles in modulating susceptibility to DE neurotoxicity.

Association Between Paraoxonase 2 Ser311Cys Polymorphism and Coronary Heart Disease Risk: A Meta-Analysis.

BackgroundThe relationship between coronary heart disease (CHD) and the paraoxonase 2 (PON2) Ser311Cys polymorphism has received much attention. We conducted a meta-analysis on the results from published case-control studies examining this relation.Material/MethodsA literature search was performed using PubMed and ISI Web of Knowledge databases until October 2015. Odds ratios (OR) and 95% confidence intervals (CI) were calculated using Stata version 11.0 software. Data were pooled using the random-effects model.ResultsNine studies were eligible for statistical analysis and included a total of 5278 participants. The results did not support an association between the Ser311Cys polymorphism and CHD in the overall populations (Asians, Caucasians, and a Hispanic mixed population) under dominant (OR 1.07; 95% CI 0.91–1.28; Pz=0.413), recessive (OR 1.19; 95% CI 0.72–1.95; Pz=0.500), homozygote (OR 1.20; 95% CI 0.71–2.03; Pz=0.489), and allelic comparison (OR 1.08; 95% CI 0.91–1.28; Pz=0.390) models. However, in subgroup analysis according to ethnicity, we found that the Ser311Cys polymorphism was associated with CHD risk in Caucasians under recessive (OR 2.08; 95% CI 1.30–3.34; Pz=0.002) and homozygote (OR 2.16; 95% CI 1.33–3.50; Pz=0.002) models. Subgroup analysis indicated no significant association of this polymorphism with CHD in either Asian or Hispanic populations.ConclusionsThe PON2 Ser311Cys polymorphism is associated with CHD risk in Caucasians, but there is no association between this polymorphism and CHD in Asians or Hispanic populations.

Paraoxonase-2 and paraoxonase-3: comparison of mRNA expressions in the placentae of unexplained intrauterine growth restricted and noncomplicated pregnancies

Objective: Recent evidence suggests that oxidative stress is involved in the pathophysiology of many human diseases. It has been demonstrated that oxidative stress is associated with intrauterine growth restriction (IUGR), and the depletion of placental antioxidant systems has been suggested as a key factor in this disease. Our aims were to explore the possible role of antioxidant paraoxonase-2 (PON2) and paraoxonase-3 (PON3) in the pathophysiology of unexplained IUGR.Methods: We have studied the expression of mRNA for PON2, PON3 in placental tissues by using RT-qPCR. Two groups, consisting of normal (n = 18) and unexplained IUGR pregnancies (n = 20) were compared. Results: Our results demonstrated that there were no significant differences in the mRNA expressions of PON2, PON3 between the two groups (p = 0.28, p = 0.90, respectively). PON2 and PON3 were down-regulated in IUGR. Antenatal steroid therapy had no effect on the expression mRNA in placentae of unexplained IUGR pregnancies compared to non-treated group.Conclusions: These results suggest that PON2, PON3 mRNA levels were not changed significantly in placentae of IUGR when compared to normal pregnant women.

Paraoxonase 2 modulates a proapoptotic function in LS174T cells in response to quorum sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone.

A mucus layer coats the gastrointestinal tract and serves as the first line of intestinal defense against infection. N-acyl-homoserine lactone (AHL) quorum-sensing molecules produced by gram-negative bacteria in the gut can influence the homeostasis of intestinal epithelium. In this study, we investigated the effects of two representative long- and short-chain AHLs, N-3-(oxododecanoyl)-homoserine lactone (C12-HSL) and N-butyryl homoserine lactone (C4-HSL), on cell viability and mucus secretion in LS174T cells. C12-HSL but not C4-HSL significantly decreased cell viability by inducing mitochondrial dysfunction and activating cell apoptosis which led to a decrease in mucin expression. Pretreatment with lipid raft disruptor (Methyl-β-cyclodextrin, MβCD) and oxidative stress inhibitor (N-acetyl-L-cysteine, NAC) slightly rescued the viability of cells damaged by C12-HSL exposure, while the paraoxonase 2 (PON2) inhibitor (Triazolo[4,3-a]quinolone, TQ416) significantly affected recovering cells viability and mucin secretion. When LS174T cells were treated with C12-HSL and TQ416 simultaneously, TQ416 showed the maximal positive effect on cells viability. However, if cells were first treated with C12-HSL for 40 mins, and then TQ46 was added, the TQ416 had no effect on cell viability. These results suggest that the C12-HSL-acid process acts at an early step to activate apoptosis as part of C12-HSL’s effect on intestinal mucus barrier function.

An improved polymerase chain reaction-restriction fragment length polymorphism assay for the detection of a PON2 gene polymorphism.

In recent research, it has been shown that there have been variants of rs12026 within the paraoxonase 2 (PON2) gene, which have been associated with cardiovascular disease, cerebrovascular disease, diabetes and other diseases. The isochizomers, such as the BsoFI enzyme, required for the detection of this polymorphism are expensive. Therefore, an improved and less expensive polymerase chain reaction (PCR)-restriction fragment length polymorphism method was established for the detection of the single-nucleotide polymorphism rs12026 in the exon 5 of chromosome 7 of the human PON2 gene using the method of amplification-created restriction site. Subsequent to assessing 302 individuals, the genotype frequencies were 68.9% for CC, 29.8% for CG and 1.3% for GG, and the allelic frequencies were 83.8% for C and 16.2% for G. The PCR results were confirmed by DNA sequencing. The χ2 test showed that the genotype and allele frequencies of PON2-148 do not deviate from Hardy-Weinberg equilibrium, and the sequences of amplified products were consistent with the sequence published in GenBank with the exception of a mismatched base.

The anti-apoptotic PON2 protein is Wnt/β-catenin-regulated and correlates with radiotherapy resistance in OSCC patients.

Aberrant Wnt signaling and control of anti-apoptotic mechanisms are pivotal features in different types of cancer to undergo cell death programs. The intracellular human enzyme Paraoxonase-2 (PON2) is known to have anti-apoptotic properties in leukemia and oral squamous cell cancer (OSCC) cells. However, the distinct regulating pathways are poorly understood. First, we present a so far unknown regulation of PON2 protein expression through the Wnt/GSK3β/β-catenin pathway in leukemia and OSCC cells. This was confirmed via in silico analysis, promoter reporter studies and treatment of multiple cell lines (K562, SCC-4, PCI-13) with different Wnt ligands/inhibitors in vitro. Ex vivo analysis of OSCC patients revealed a correlation between PON2 and β-catenin expression in tumor tissue. Higher PON2 expression in OSCC is associated with relapse independently of treatment (e.g. surgery/radio-/chemotherapy). These results emphasize the clinical impact of the newly described regulation of PON2 through Wnt/GSK3β/β-catenin. More importantly, the study revealed the fundamental finding of an overall Wnt/GSK3β/β-catenin dependent regulation of PON2 in different cancers, which was confirmed by systematic and multimethodological approaches. Thus, the herein presented mechanistic insight contributes to a better understanding of tumor specific escape from cell death strategies and suggests PON2 as a new potential biomarker for therapy resistance or as a prognostic tumor marker.

Aqueous or lipid components of atherosclerotic lesion increase macrophage oxidation and lipid accumulation

Introduction and objectiveUnderstanding the interactions among atherosclerotic plaque components and arterial macrophages, is essential for elucidating the mechanisms involved in the development of atherosclerosis. We assessed the effects of lesion extracts on macrophages. MethodsMouse peritoneal macrophages from atherosclerotic normoglycemic or hyperglycemic apoE−/− mice were incubated with aortic aqueous or with aortic lipidic extracts (mAAE or mALE) derived from these mice. In parallel, J774A.1 cultured macrophages were incubated with increasing concentrations of extracts prepared from human carotid lesions: polar lesion aqueous extract (hLAE), nonpolar lesion lipid extract (hLLE), or with their combination. In all the above systems we performed analyses of macrophage oxidative status, cholesterol, and triglyceride metabolism. ResultsAqueous or lipid extracts from either mice aorta or from human carotid lesions significantly increased macrophage oxidative stress as determined by reactive oxygen species (ROS) analysis. In parallel, a compensatory increase in the cellular antioxidant paraoxonase2 (PON2) activity and in macrophage glutathione content were observed following incubation with all extracts. Macrophage triglyceride mass and triglyceride biosynthesis rate were both significantly increased following treatment with the lipid extracts, secondary to upregulation of DGAT1. All extracts decreased cholesterol biosynthesis rate, through downregulation of HMGCR, the rate limiting enzyme in cholesterol biosynthesis. The combination of the human lesion extracts had the most significant effects. ConclusionThe present study demonstrates that atherosclerotic plaque constituents enhance macrophage cellular oxidative stress, and accumulation of cholesterol and triglycerides, as shown in both in vivo and in vitro model systems.

Enhanced Clearance of Pseudomonas aeruginosa by Peroxisome Proliferator-Activated Receptor Gamma.

The pathogenic profile of Pseudomonas aeruginosa is related to its ability to secrete a variety of virulence factors. Quorum sensing (QS) is a mechanism wherein small diffusible molecules, specifically acyl-homoserine lactones, are produced by P. aeruginosa to promote virulence. We show here that macrophage clearance of P. aeruginosa (PAO1) is enhanced by activation of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARγ). Macrophages treated with a PPARγ agonist (pioglitazone) showed enhanced phagocytosis and bacterial killing of PAO1. It is known that PAO1 QS molecules are inactivated by PON-2. QS molecules are also known to inhibit activation of PPARγ by competitively binding PPARγ receptors. In accord with this observation, we found that infection of macrophages with PAO1 inhibited expression of PPARγ and PON-2. Mechanistically, we show that PPARγ induces macrophage paraoxonase 2 (PON-2), an enzyme that degrades QS molecules produced by P. aeruginosa Gene silencing studies confirmed that enhanced clearance of PAO1 in macrophages by PPARγ is PON-2 dependent. Further, we show that PPARγ agonists also enhance clearance of P. aeruginosa from lungs of mice infected with PAO1. Together, these data demonstrate that P. aeruginosa impairs the ability of host cells to mount an immune response by inhibiting PPARγ through secretion of QS molecules. These studies define a novel mechanism by which PPARγ contributes to the host immunoprotective effects during bacterial infection and suggest a role for PPARγ immunotherapy for P. aeruginosa infections.

Developmental expression of paraoxonase 2

Paraoxonase 2 (PON2) is a member of the paraoxonase gene family also comprising PON1 and PON3. PON2 functions as a lactonase and exhibits anti-bacterial as well as antioxidant properties. At the cellular level, PON2 localizes to the mitochondrial and endoplasmic reticulum membranes where it scavenges reactive oxygen species. PON2 is of particular interest as it is the only paraoxonase expressed in brain tissue and appears to play a critical role in mitigating oxidative stress in the brain. The aim of this study was to investigate the expression of PON2 at the protein and mRNA level in the brain and liver of mice through development to identify potential age windows of susceptibility to oxidative stress, as well as to compare expression of hepatic PON2 to expression of PON1 and PON3. Overall, PON2 expression in the brain was lower in neonatal mice and increased with age up to postnatal day (PND) 21, with a significant decrease observed at PND 30 and 60. In contrast, the liver showed continuously increasing levels of PON2 with age, similar to the patterns of PON1 and PON3. PON2 protein levels were also investigated in brain samples from non-human primates, with PON2 increasing with age up to the infant stage and decreasing at the juvenile stage, mirroring the results observed in the mouse brain. These variable expression levels of PON2 suggest that neonatal and young adult animals may be more susceptible to neurological insult by oxidants due to lower levels of PON2 in the brain.

Identification of Paraoxonase‐2 as a Novel Modulator of the Epithelial Na+ Channel

Paraoxonase‐2 (PON‐2) is a membrane‐bound lactonase with unique anti‐oxidative and anti‐atherosclerotic properties, and thus has been associated with cardiovascular diseases. PON‐2 shares key structural elements with MEC‐6, an essential component of the C. elegans mechanosensitive ion channel complex required for the worm's touch response. At the core of this channel complex, MEC‐6 interacts with two pore‐forming subunits, MEC‐4 and MEC‐10 and enhances channel activity. MEC‐4 and MEC‐10 belong to the same family as the epithelial Na+ channel (ENaC), a key mediator of Na+ uptake in the aldosterone sensitive distal nephron (ASDN). We therefore examined PON‐2 expression in the ASDN, and immunofluorescence microscopy indicated tubular‐specific expression of PON‐2 that included aquaporin 2‐expressing cells. When PON‐2 was co‐expressed with individual ENaC subunits in HEK293 cells, PON‐2 and each of the three ENaC subunits could be co‐immunoprecipitated. In contrast, PON‐2 co‐immunoprecipitated only the α subunit of ENaC when all three subunits were expressed. When PON‐2 was co‐expressed with ENaC in Xenopus oocytes, both whole cell Na+ currents and ENaC surface expression were reduced. This inhibitory effect was ENaC‐specific, as PON‐2 had no effect on ROMK activity. However, the PON‐2 inhibitory effect was lost with a PON‐2 mutant (D268A) that abrogates the lactonase activity of PON‐2, likely as a result of interfering with calcium binding. Together, our results indicate that PON‐2 interacts with ENaC and modulates channel expression.

PON2 and ATP2B2 gene polymorphisms with noise-induced hearing loss.

Noise-induced hearing loss (NIHL) is a complex disease induced by a combination of genetic and environmental factors. Paraoxonase2 (PON2) gene involved in the regulation of reactive oxygen species, and affecting the vulnerability of cochlea to NIHL, and ATPase, calcium-transporting, plasma membrane 2 (ATP2B2) gene which encodes plasma membrane calcium-transporting ATPase isoform 2 (PMCA2) are the candidate genes relating to the attack of NIHL. In this study, we investigated whether ATP2B2 and PON2 polymorphisms were associated with NIHL in Chinese of Han nationality population. We performed a case-control study between six single nucleotide polymorphisms (SNPs) (rs1719571, rs3209637 and rs4327369 within ATP2B2, rs12026, rs7785846 and rs12704796 within PON2) and NIHL in 454 subjects. All the SNPs were genotypes, using the TaqMan MGB probe assay. Odds ratios (ORs) were calculated with 95% confidence intervals (95% CIs) with logistic regression analysis to test the level of association for SNPs. In our study, 221 subjects with hearing loss and 233 subjects without hearing loss were recruited. The frequencies of the CG and CG + GG genotype of rs12026 (PON2) conferred risk factors for NIHL with adjusted OR values of 2.62 (95% CI, 1.69-4.06) and 2.48 (95% CI, 1.63-3.78), respectively. This kind of significance was also found at locus rs7785846, where genotypes CT and CT + TT were the risk types, with adjusted ORs of 2.52 (95% CI, 1.62-3.93) and 2.35 (95% CI, 1.54-3.58), respectively. We performed stratified analysis per noise exposure level, when it came to rs7785846 and rs12026 in the >92 dB(A) noise exposure group, the subjects who carried heterozygote were of significantly (P<0.01) higher susceptibility to NIHL than homozygote carriers. By contrast, no significantly higher risk was found for any rs12704796 genotypes or any genotypes in ATP2B2 (P>0.05), which may suggest that these SNPs did not have significant effects on noise susceptibility across noise exposure. Our research suggested that PON2 might play a role in the etiology of NIHL in Chinese of Han nationality population.