Journal of Lipids

Treadmill Exercise Training Modulates Hepatic Cholesterol Metabolism and Circulating PCSK9 Concentration in High-Fat-Fed Mice

Shin Wen, Kavita S. Jadhav, David L. Williamson, and Todd C. Rideout — Wed, 19 Jun 2013 08:55:14 +0000

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a novel biomarker of LDL clearance and a therapeutic target of cardiovascular disease. We examined the effects of aerobic exercise training in modulating PCSK9 abundance and hepatic sterol regulation in high-fat-fed C57BL/6 mice. Mice () were assigned to a low-fat (LF), high-fat (HF), or an HF with exercise (HF + EX) group for 8 weeks. The HF + EX group was progressively trained 5 days/week on a motorized treadmill. The HF + EX group was protected against body weight (BW) gain and diet-induced dyslipidemia compared with the HF group. The HF + EX group demonstrated an increase in hepatic PCSK9 mRNA (1.9-fold of HF control, ) and a reduction in plasma PCSK9 (14%) compared with the HF group. Compared with HF mice, HF + EX mice demonstrated reduced hepatic cholesterol (14%) and increased () nuclear SREBP2 protein (1.8-fold of HF group) and LDLr mRNA (1.4-fold of HF group). Plasma PCSK9 concentrations correlated positively with plasma non-HDL-C (, ). Results suggest that treadmill exercise reduces non-HDL cholesterol and differentially modulates hepatic and blood PCSK9 abundance in HF-fed C57BL/6 mice.

5-Lipoxygenase-Activating Protein as a Modulator of Olanzapine-Induced Lipid Accumulation in Adipocyte

Svetlana Dzitoyeva, Hu Chen, and Hari Manev — Sat, 25 May 2013 15:55:30 +0000

Experiments were performed in 3T3-L1 preadipocytes differentiated in vitro into adipocytes. Cells were treated with olanzapine and a 5-lipoxygenase (5-LOX) activating protein (FLAP) inhibitor MK-886. Lipid content was measured using an Oil Red O assay; 5-LOX and FLAP mRNA content was measured using quantitative real-time PCR; the corresponding protein contents were measured using quantitative Western blot assay. Olanzapine did not affect the cell content of 5-LOX mRNA and protein; it decreased FLAP mRNA and protein content at day five but not 24 hours after olanzapine addition. In the absence of MK-886, low concentrations of olanzapine increased lipid content only slightly, whereas a 56% increase was induced by 50 μM olanzapine. A 5-day cotreatment with 10 μM MK-886 potentiated the lipid increasing action of low concentrations of olanzapine. In contrast, in the presence of 50 μM olanzapine nanomolar and low micromolar concentrations of MK-886 reduced lipid content. These data suggest that FLAP system in adipocytes is affected by olanzapine and that it may modify how these cells respond to the second-generation antipsychotic drugs (SGADs). Clinical studies could evaluate whether the FLAP/5-LOX system could play a role in setting a variable individual susceptibility to the metabolic side effects of SGADs.

The Multifaceted Effects of Omega-3 Polyunsaturated Fatty Acids on the Hallmarks of Cancer

Thu, 16 May 2013 14:12:46 +0000

Omega-3 polyunsaturated fatty acids, in particular eicosapentaenoic acid, and docosahexaenoic acid have been shown to have multiple beneficial antitumour actions that affect the essential alterations that dictate malignant growth. In this review we explore the putative mechanisms of action of omega-3 polyunsaturated fatty acid in cancer protection in relation to self-sufficiency in growth signals, insensitivity to growth-inhibitory signals, apoptosis, limitless replicative potential, sustained angiogenesis, and tissue invasion, and how these will hopefully translate from bench to bedside.

Trans-10, cis 12-Conjugated Linoleic Acid-Induced Milk Fat Depression Is Associated with Inhibition of PPAR Signaling and Inflammation in Murine Mammary Tissue

Tue, 14 May 2013 14:48:04 +0000

Exogenous trans-10, cis-12-CLA (CLA) reduces lipid synthesis in murine adipose and mammary (MG) tissues. However, genomewide alterations in MG and liver (LIV) associated with dietary CLA during lactation remain unknown. We fed mice (/diet) control or control + trans-10, cis-12-CLA (37 mg/day) between d 6 and d 10 postpartum. The 35,302 annotated murine exonic evidence-based oligo (MEEBO) microarray and quantitative RT-PCR were used for transcript profiling. Milk fat concentration was 44% lower on d 10 versus d 6 due to CLA. The CLA diet resulted in differential expression of 1,496 genes. Bioinformatics analyses underscored that a major effect of CLA on MG encompassed alterations in cellular signaling pathways and phospholipid species biosynthesis. Dietary CLA induced genes related to ER stress (Xbp1), apoptosis (Bcl2), and inflammation (Orm1, Saa2, and Cp). It also induced marked inhibition of PPARγ signaling, including downregulation of Pparg and Srebf1 and several lipogenic target genes (Scd, Fasn, and Gpam). In LIV, CLA induced hepatic steatosis probably through perturbations in the mitochondrial functions and induction of ER stress. Overall, results from this study underscored the role of PPARγ signaling on mammary lipogenic target regulation. The proinflammatory effect due to CLA could be related to inhibition of PPARγ signaling.

Roles of Fatty Acid Oversupply and Impaired Oxidation in Lipid Accumulation in Tissues of Obese Rats

Mon, 13 May 2013 14:13:45 +0000

To test the roles of lipid oversupply versus oxidation in causing tissue lipid accumulation associated with insulin resistance/obesity, we studied in vivo fatty acid (FA) metabolism in obese (Obese) and lean (Lean) Zucker rats. Indices of local FA utilization and storage were calculated using the partially metabolizable [9,10-3H]-(R)-2-bromopalmitate (3H-R-BrP) and [U-14C]-palmitate (14C-P) FA tracers, respectively. Whole-body FA appearance () was estimated from plasma 14C-P kinetics. Whole-body FA oxidation rate () was assessed using 3H2O production from 3H-palmitate infusion, and tissue FA oxidative capacity was evaluated ex vivo. In the basal fasting state Obese had markedly elevated FA levels and , associated with elevated FA utilization and storage in most tissues. Estimated rates of muscle FA oxidation were not lower in obese rats and were similarly enhanced by contraction in both lean and obese groups. At comparable levels of FA availability, achieved by nicotinic acid, was lower in Obese than Lean. In Obese rats, FA oxidative capacity was 35% higher than that in Lean in skeletal muscle, 67% lower in brown fat and comparable in other organs. In conclusion, lipid accumulation in non-adipose tissues of obese Zucker rats appears to result largely from systemic FA oversupply.

Modification of Phospholipid Bilayers Induced by Sulfurated Naphthoquinones

Claudio Di Vitta, Liliana Marzorati, and Sérgio S. Funari — Sat, 30 Mar 2013 14:14:56 +0000

New thionaphthoquinones and their hydroxyl derivatives, bearing alkyl side chains that match the phospholipids POPC and POPE, were synthesized in order to investigate their interactions with lipids. It was observed that, in general, these additives destabilize the lipid bilayer and induce less organized structures with higher curvature, in particular the induction of an hexagonal phase on aqueous POPC mixtures. Moreover, cubic phases, not normally observed in the pure lipids when fully hydrated, were detected. Coexistence of lamellar phases was interpreted as a consequence of microsegregation of the components in the mixtures. These results are in line with previous observations on the effect of structurally similar (hydro)quinones in phase behavior of these lipids.

PPARγ Networks in Cell Signaling: Update and Impact of Cyclic Phosphatidic Acid

Tamotsu Tsukahara — Thu, 07 Feb 2013 11:27:49 +0000

Lysophospholipid (LPL) has long been recognized as a membrane phospholipid metabolite. Recently, however, the LPL has emerged as a candidate for diagnostic and pharmacological interest. LPLs include lysophosphatidic acid (LPA), alkyl glycerol phosphate (AGP), cyclic phosphatidic acid (cPA), and sphingosine-1-phosphate (S1P). These biologically active lipid mediators serve to promote a variety of responses that include cell proliferation, migration, and survival. These LPL-related responses are mediated by cell surface G-protein-coupled receptors and also intracellular receptor peroxisome proliferator-activated receptor gamma (PPARγ). In this paper, we focus mainly on the most recent findings regarding the biological function of nuclear receptor-mediated lysophospholipid signaling in mammalian systems, specifically as they relate to health and diseases. Also, we will briefly review the biology of PPARγ and then provide an update of lysophospholipids PPARγ ligands that are under investigation as a therapeutic compound and which are targets of PPARγ relevant to diseases.

The Impairment of Macrophage-to-Feces Reverse Cholesterol Transport during Inflammation Does Not Depend on Serum Amyloid A

Wed, 30 Jan 2013 13:35:00 +0000

Studies suggest that inflammation impairs reverse cholesterol transport (RCT). We investigated whether serum amyloid A (SAA) contributes to this impairment using an established macrophage-to-feces RCT model. Wild-type (WT) mice and mice deficient in SAA1.1 and SAA2.1 (SAAKO) were injected intraperitoneally with 3H-cholesterol-labeled J774 macrophages 4 hr after administration of LPS or buffered saline. 3H-cholesterol in plasma 4 hr after macrophage injection was significantly reduced in both WT and SAAKO mice injected with LPS, but this was not associated with a reduced capacity of serum from LPS-injected mice to promote macrophage cholesterol efflux in vitro. Hepatic accumulation of 3H-cholesterol was unaltered in either WT or SAAKO mice by LPS treatment. Radioactivity present in bile and feces of LPS-injected WT mice 24 hr after macrophage injection was reduced by 36% and 80% , respectively. In contrast, in SAAKO mice, LPS did not significantly reduce macrophage-derived 3H-cholesterol in bile, and fecal excretion was reduced by only 45% . Injection of cholesterol-loaded allogeneic J774 cells, but not syngeneic bone-marrow-derived macrophages, transiently induced SAA in C57BL/6 mice. Our study confirms reports that acute inflammation impairs steps in the RCT pathway and establishes that SAA plays only a minor role in this impairment.

High-Density Lipoproteins and the Immune System

Hidesuke Kaji — Wed, 30 Jan 2013 13:18:17 +0000

High-density lipoprotein (HDL) plays a major role in vasodilation and in the reduction of low-density lipoprotein (LDL) oxidation, inflammation, apoptosis, thrombosis, and infection; however, HDL is now less functional in these roles under certain conditions. This paper focuses on HDL, its anti-inflammation behavior, and the mechanisms by which HDL interacts with components of the innate and adaptive immune systems. Genome-wide association studies (GWAS) and proteomic studies have elucidated important molecules involved in the interaction between HDL and the immune system. An understanding of these mechanisms is expected to be useful for the prevention and treatment of chronic inflammation due to metabolic syndrome, atherosclerosis, or various autoimmune diseases.

Molecular Characterization of Lipopolysaccharide Binding to Human α-1-Acid Glycoprotein

Thu, 20 Dec 2012 09:22:15 +0000

The ability of AGP to bind circulating lipopolysaccharide (LPS) in plasma is believed to help reduce the proinflammatory effect of bacterial lipid A molecules. Here, for the first time we have characterized human AGP binding characteristics of the LPS from a number of pathogenic Gram-negative bacteria: Escherichia coli, Salmonella typhimurium, Klebsiella pneumonia, Pseudomonas aeruginosa, and Serratia marcescens. The binding affinity and structure activity relationships (SAR) of the AGP-LPS interactions were characterized by surface plasma resonance (SPR). In order to dissect the contribution of the lipid A, core oligosaccharide and O-antigen polysaccharide components of LPS, the AGP binding affinity of LPS from smooth strains, were compared to lipid A, Kdo2-lipid A, , , and rough LPS mutants. The SAR analysis enabled by the binding data suggested that, in addition to the important role played by the lipid A and core components of LPS, it is predominately the unique species- and strain-specific carbohydrate structure of the O-antigen polysaccharide that largely determines the binding affinity for AGP. Together, these data are consistent with the role of AGP in the binding and transport of LPS in plasma during acute-phase inflammatory responses to invading Gram-negative bacteria.

Sphingosine 1-Phosphate Distribution in Human Plasma: Associations with Lipid Profiles

Samar M. Hammad, Mohammed M. Al Gadban, Andrea J. Semler, and Richard L. Klein — Thu, 08 Nov 2012 15:17:48 +0000

The physiological significance of sphingosine 1-phosphate (S1P) transport in blood has been debated. We have recently reported a comprehensive sphingolipid profile in human plasma and lipoprotein particles (VLDL, LDL, and HDL) using HPLC-MS/MS (Hammad et al., 2010). We now determined the relative concentrations of sphingolipids including S1P in the plasma subfraction containing lipoproteins compared to those in the remaining plasma proteins. Sphingomyelin and ceramide were predominantly recovered in the lipoprotein-containing fraction. Total plasma S1P concentration was positively correlated with S1P concentration in the protein-containing fraction, but not with S1P concentration in the lipoprotein-containing fraction. The percentage of S1P transported in plasma lipoproteins was positively correlated with HDL cholesterol (HDL-C) concentration; however, S1P transport in lipoproteins was not limited by the concentration of HDL-C in the individual subject. Thus, different plasma pools of S1P may have different contributions to S1P signaling in health and disease.

Hepatic Metabolic, Inflammatory, and Stress-Related Gene Expression in Growing Mice Consuming a Low Dose of Trans-10, cis-12-Conjugated Linoleic Acid

Jing Li, Srikant Viswanadha, and Juan J. Loor — Sun, 02 Sep 2012 14:26:08 +0000

Dietary trans-10, cis-12-conjugated linoleic acid (trans-10, cis-12-CLA) fed to obese and nonobese rodents reduces body fat but leads to greater liver mass due to steatosis. The molecular mechanisms accompanying such responses remain largely unknown. Our study investigated the effects of chronic low trans-10, cis-12-CLA supplementation on hepatic expression of 39 genes related to metabolism, inflammation, and stress in growing mice. Feeding a diet supplemented with 0.3% trans-10, cis-12-CLA (wt/wt basis) for 6 weeks increased liver mass and concentration of long-chain fatty acids (LCFAs) in liver, while adipose tissue mass decreased markedly. These changes were accompanied by greater expression of genes involved in LCFA uptake (Cd36), lipogenesis, and triacylglycerol synthesis (Acaca, Gpam, Scd, Pck1, Plin2). Expression of these genes was in line with upregulation of the lipogenic transcription factor Srebf1. Unlike previous studies where higher >0.50% of the diet) doses of trans-10, cis-12-CLA were fed, we found greater expression of genes associated with VLDL assembly/secretion (Mttp, Cideb), ketogenesis (Hmgcs2, Bdh1), and LCFA oxidation (Acox1, Pdk4) in response to trans-10, cis-12-CLA. Dietary CLA, however, did not affect inflammation- and stress-related genes. Results suggested that a chronic low dose of dietary CLA increases liver mass and lipid accumulation due to activation of lipogenesis and insufficient induction of LCFA oxidation and VLDL assembly/secretion.

Dietary Omega-3 Fatty Acids Do Not Change Resistance of Rat Brain or Liver Mitochondria to Ca2+ and/or Prooxidants

Mon, 27 Aug 2012 13:22:36 +0000

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) block apoptotic neuronal cell death and are strongly neuroprotective in acute and chronic neurodegeneration. Theoretical considerations, indirect data, and consideration of parsimony lead to the hypothesis that modulation of mitochondrial pathway(s) underlies at least some of the neuroprotective effects of n-3 PUFAs. We therefore systematically tested this hypothesis on healthy male FBFN1 rats fed for four weeks with isocaloric, 10% fat-containing diets supplemented with 1, 3, or 10% fish oil (FO). High resolution mass spectrometric analysis confirmed expected diet-driven increases in docosahexaenoic acid (DHA, 22:6, n-3) and eicosapentaenoic acid (EPA, 20:5, n-3) in sera, liver and nonsynaptosomal brain mitochondria. We further evaluated the resistance of brain and liver mitochondria to Ca2+ overload and prooxidants. Under these conditions, neither mitochondrial resistance to Ca2+ overload and prooxidants nor mitochondrial physiology is altered by diet, despite the expected incorporation of DHA and EPA in mitochondrial membranes and plasma. Collectively, the data eliminate one of the previously proposed mechanism(s) that n-3 PUFA induced augmentation of mitochondrial resistance to the oxidant/calcium-driven dysfunction. These data furthermore allow us to define a specific series of follow-up experiments to test related hypotheses about the effect of n-3 PUFAs on brain mitochondria.

Fatty Acid Composition of Phospholipids and in the Central and External Positions of Triacylglycerol in Muscle and Subcutaneous Fat of Beef Steers Fed Diets Supplemented with Oil Containing n6 and n3 Fatty Acids While Undergoing One of Three 48 h Feed Withdrawal Treatments

C. Margetak, G. Travis, T. Entz, P. S. Mir, S. Wei, and M. V. Dodson — Sun, 29 Jul 2012 09:51:24 +0000

This study was designed to determine the effects of dietary oil and feed withdrawal treatments on fatty acid composition of phospholipids of triacylglycerol in pars costalis diaphragmatis muscle and subcutaneous fat from the brisket. A 2 × 3 factorial experiment was conducted with crossbred steers with an initial body weight of 280.5 ± 5.8 kg. Steers were fed either a control or an oil containing diet where 5% of the control diet was replaced with an equal mixture sunflower and flax oil while undergoing one of three feed withdrawal treatments: no withdrawal, a single 48 h withdrawal before initiation of fattening at one year of age, or 48 h withdrawal at 8 wk intervals from weaning to initiation of fattening. At time of processing samples of muscle and fat were obtained and analyzed to determine fatty acid composition. Disproportionate distribution of the fatty acids was observed by diet, feed withdrawal regimen and whether the sample was from muscle or fat. Differences are discussed in detail, and our data suggests a special function for the fatty acids that accumulate in specific positions of the triacylglycerol due to treatment.

A Nonradioactive Fluorimetric SPE-Based Ceramide Kinase Assay Using NBD-C6-Ceramide

Helena Van Overloop, Gerd Van der Hoeven, and Paul P. Van Veldhoven — Thu, 26 Jul 2012 10:46:29 +0000

Ceramide kinase (CERK) has been implicated in important cellular processes such as inflammation and apoptosis. Its activity is usually measured using radiolabeled ceramide or [γ-32P]-ATP, followed by extraction, thin-layer chromatography, and detection of the formed labeled ceramide-1-phosphate. To eliminate the use of radioactivity, we developed similarly but independently from the approach by Don and Rosen (2008), a fluorescence-based ceramide kinase assay, using N-[7-(4-nitrobenz-2-oxa-1,3-diazole)]-6-aminohexanoyl-sphingenine (NBD-C6-ceramide) as substrate. Its 𝐾𝑚 value (4 μM) was comparable to that of N-hexanoyl-sphingenine (C6-ceramide). The produced fluorescent NBD-C6-ceramide-1-phosphate was captured by means of solid-phase extraction on an aminopropyl phase, resulting in a fast and sensitive CERK measurement. By performing this assay in a 96-well format, it is also suitable for high-throughput screening (HTS) to search for CERK modulators. A limited screen revealed that some protein kinase inhibitors (e.g., U-0126; IC50 4 μM) and ceramide analogues (e.g., fenretinide, AMG-9810; IC50 1.1 μM) affect CERK in vitro.

The Phospholipid Profile of Mycoplasmas

Jonathan D. Kornspan and Shlomo Rottem — Sun, 15 Jul 2012 10:18:04 +0000

The de novo synthesized polar lipids of Mycoplasma species are rather simple, comprising primarily of the acidic glycerophospholipids PG and CL. In addition, when grown in a medium containing serum, significant amounts of PC and SPM are incorporated into the mycoplasma cell membrane although these lipids are very uncommon in wall-covered bacteria. The exogenous lipids are either incorporated unchanged or the PC incorporated is modified by a deacylation-acylation enzymatic cycle to form disaturated PC. Although their small genome, in some Mycoplasma species, other genes involved in lipid biosynthesis were detected, resulting in the synthesis of a variety of glycolipis, phosphoglycolipids and ether lipids. We suggest that analyses and comparisons of mycoplasma polar lipids may serve as a novel and useful tool for classification. Nonetheless, to evaluate the importance of polar lipids in mycoplasma, further systematic and extensive studies on more Mycoplasma species are needed. While studies are needed to elucidate the role of lipids in the mechanisms governing the interaction of mycoplasmas with host eukaryotic cells, the finding that a terminal phosphocholine containing glycolipids of M. fermentans serves both as a major immune determinants and as a trigger of the inflammatory responses, and the findings that the fusogenicity of M. fermentans with host cells is markedly stimulated by lyso-ether lipids, are important steps toward understanding the molecular mechanisms of M. fermentans pathogenicity.

Use of the Signature Fatty Acid 16:1𝝎5 as a Tool to Determine the Distribution of Arbuscular Mycorrhizal Fungi in Soil

Christopher Ngosong, Elke Gabriel, and Liliane Ruess — Thu, 05 Jul 2012 09:48:26 +0000

Biomass estimation of arbuscular mycorrhiza (AM) fungi, widespread plant root symbionts, commonly employs lipid biomarkers, predominantly the fatty acid 16:1ω5. We briefly reviewed the application of this signature fatty acid, followed by a case study comparing biochemical markers with microscopic techniques in an arable soil following a change to AM non-host plants after 27 years of continuous host crops, that is, two successive cropping seasons with wheat followed by amaranth. After switching to the non-host amaranth, spore biomass estimated by the neutral lipid fatty acid (NLFA) 16:1ω5 decreased to almost nil, whereas microscopic spore counts decreased by about 50% only. In contrast, AM hyphal biomass assessed by the phospholipid (PLFA) 16:1ω5 was greater under amaranth than wheat. The application of PLFA 16:1ω5 as biomarker was hampered by background level derived from bacteria, and further enhanced by its incorporation from degrading spores used as microbial resource. Meanwhile, biochemical and morphological assessments showed negative correlation for spores and none for hyphal biomass. In conclusion, the NLFA 16:1ω5 appears to be a feasible indicator for AM fungi of the Glomales group in the complex field soils, whereas the use of PLFA 16:1ω5 for hyphae is unsuitable and should be restricted to controlled laboratory studies.

The Effect of Egg Consumption in Hyperlipidemic Subjects during Treatment with Lipid-Lowering Drugs

Sun, 24 Jun 2012 08:20:35 +0000

Background. Limiting egg consumption to avoid high cholesterolemia is recommended to reduce risk of cardiovascular disease. However, recent evidences suggest that cholesterol from diet has limited influence on serum cholesterol. Objective. To assess the effect of egg consumption on lipid profiles in hyperlipidemic adults treated with lipid-lowering drugs. Material and Method. Sixty hyperlipidemic subjects, mean age of 61 years, who had been treated with lipid-lowering drugs. Every subject was assigned to consume additional 3 eggs per day with their regular diet for 12 weeks. Measurements for lipid profiles and body compositions were performed. Results. An additional consumption of 3 eggs per day for 12 weeks increased HDL-cholesterol by 2.46±6.81 mg/dL (𝑃<0.01) and decreased LDL-cholesterol to HDL-cholesterol ratio by 0.13±0.46 (𝑃<0.05). No significant changes were found in other lipid profiles. Body weight and body mass index were significantly increased at 12th week by 0.52±1.83 kg and 0.31±0.99 kg/m2, respectively (𝑃<0.05). Conclusion. In hyperlipidemic adults who were treated with lipid-lowering drugs, the consumption of additional 3 eggs per day to their regular diet will increase the level of HDL-cholesterol and decrease the ratio of LDL-cholesterol to HDL-cholesterol.

Supercritical SC-CO2 and Soxhlet n-Hexane Extract of Tunisian Opuntia ficus indica Seeds and Fatty Acids Analysis

Sun, 17 Jun 2012 10:39:32 +0000

The fatty acids profiles of Tunisian Opuntia ficus indica seeds (spiny and thornless form) were investigated. Results of supercritical carbon dioxide (SC-CO2) and soxhlet n-hexane extract were compared. Quantitatively, the better yield was obtained through soxhlet n-hexane: 10.32% (spiny) and 8.91% (thornless) against 3.4% (spiny) and 1.94% (thornless) by SC-CO2 extract (T = 40°C, 𝑃=180 bar, time = 135 mn, CO2 flow rate = 15 mL·s−1). Qualitatively, the main fatty acids components were the same for the two types of extraction. Linoleic acid was the major compound, SC-CO2: 57.60% (spiny), 59.98% (thornless), soxhlet n-hexane: 57.54% (spiny), 60.66% (thornless), followed by oleic acid, SC-CO2: 22.31% (spiny), 22.40% (thornless), soxhlet n-hexane: 25.28% (spiny), 20.58% (thornless) and palmitic acid, SC-CO2: 14.3% (spiny), 12.92% (thornless), soxhlet n-hexane: 11.33% (spiny), 13.08% (thornless). The SC-CO2 profiles fatty acids showed a richness with other minority compounds such as C20:1, C20:2, and C22.The seeds oil was highly unsaturated (US = 4.44–5.25), and the rising temperatures donot affect the selectivity of fatty acids extract by SC-CO2: US = 4.44 (T = 40°C) and 4.13 (T = 70°C).

Introduction to Paraoxonases

Michael Aviram — Tue, 12 Jun 2012 08:43:31 +0000

Solubilization and Humanization of Paraoxonase-1

Thu, 07 Jun 2012 15:40:19 +0000

Paraoxonase-1 (PON1) is a serum protein, the activity of which is related to susceptibility to cardiovascular disease and intoxication by organophosphorus (OP) compounds. It may also be involved in innate immunity, and it is a possible lead molecule in the development of a catalytic bioscavenger of OP pesticides and nerve agents. Human PON1 expressed in E. coli is mostly found in the insoluble fraction, which motivated the engineering of soluble variants, such as G2E6, with more than 50 mutations from huPON1. We examined the effect on the solubility, activity, and stability of three sets of mutations designed to solubilize huPON1 with fewer overall changes: deletion of the N-terminal leader, polar mutations in the putative HDL binding site, and selection of the subset of residues that became more polar in going from huPON1 to G2E6. All three sets of mutations increase the solubility of huPON1; the HDL-binding mutant has the largest effect on solubility, but it also decreases the activity and stability the most. Based on the G2E6 polar mutations, we “humanized” an engineered variant of PON1 with high activity against cyclosarin (GF) and found that it was still very active against GF with much greater similarity to the human sequence.

Paraoxonase 1 Phenotype and Mass in South Asian versus Caucasian Renal Transplant Recipients

Sun, 03 Jun 2012 15:23:27 +0000

South Asian renal transplant recipients have a higher incidence of cardiovascular disease compared with Caucasian renal transplant recipients. We carried out a study to determine whether paraoxonase 1, a novel biomarker for cardiovascular risk, was decreased in South Asian compared with Caucasian renal transplant recipients. Subjects were matched two to one on the basis of age and sex for a total of 129 subjects. Paraoxonase 1 was measured by mass, arylesterase activity, and two-substrate phenotype assay. Comparisons were made by using a matched design. The frequency of PON1 QQ, QR and RR phenotype was 56%, 37%, and 7% for Caucasian subjects versus 35%, 44%, and 21% for South Asian subjects (𝜒2=7.72, 𝑃=0.02). PON1 mass and arylesterase activity were not significantly different between South Asian and Caucasian subjects. PON1 mass was significantly associated with PON1 phenotype (𝑃=0.0001), HDL cholesterol (𝑃=0.009), LDL cholesterol (𝑃=0.02), and diabetes status (𝑃<0.05). Arylesterase activity was only associated with HDL cholesterol (𝑃=0.003). Thus the frequency of the PON1 RR phenotype was higher and that of the QQ phenotype was lower in South Asian versus Caucasian renal transplant recipients. However, ethnicity was not a significant factor as a determinant of PON1 mass or arylesterase activity, with or without analysis including PON1 phenotype. The two-substrate method for determining PON1 phenotype may be of value for future studies of cardiovascular complications in renal transplant recipients.

Increased Levels of Human Carotid Lesion Linoleic Acid Hydroperoxide in Symptomatic and Asymptomatic Patients Is Inversely Correlated with Serum HDL and Paraoxonase 1 Activity

Tue, 29 May 2012 09:45:23 +0000

Human carotid plaque components interact directly with circulating blood elements and thus they might affect each other. We determined plaque paraoxonase1 (PON1) hydrolytic-catalytic activity and compared plaque and blood levels of lipids, HDL, PON1, and HbA1c, as well as plaque-oxidized lipids in symptomatic and asymptomatic patients. Human carotid plaques were obtained from symptomatic and asymptomatic patients undergoing routine endarterectomy, and the lesions were ground and extracted for PON activity and lipid content determinations. Plaque PONs preserved paraoxonase, arylesterase, and lactonase activities. The PON1-specific inhibitor 2-hydroxyquinoline almost completely inhibited paraoxonase and lactonase activities, while only moderately inhibiting arylesterase activity. Oxysterol and triglyceride levels in plaques from symptomatic and asymptomatic patients did not differ significantly, but plaques from symptomatic patients had significantly higher (135%) linoleic acid hydroperoxide (LA-13OOH) levels. Their serum PON1 activity, cholesterol and triglyceride levels did not differ significantly, but symptomatic patients had significantly lower (28%) serum HDL levels and higher (18%) HbA1c levels. Thus LA-13OOH, a major atherogenic plaque element, showed significant negative correlations with serum PON1 activity and HDL levels, and a positive correlation with the prodiabetic atherogenic HbA1c. Plaque PON1 retains its activity and may decrease plaque atherogenicity by reducing specific oxidized lipids (e.g., LA-13OOH). The inverse correlation between plaque LA-13OOH level and serum HDL level and PON1 activity suggests a role for serum HDL and PON1 in LA-13OOH accumulation.

Additional Common Polymorphisms in the PON Gene Cluster Predict PON1 Activity but Not Vascular Disease

Tue, 22 May 2012 14:38:15 +0000

Background. Paraoxonase 1 (PON1) enzymatic activity has been consistently predictive of cardiovascular disease, while the genotypes at the four functional polymorphisms at PON1 have not. The goal of this study was to identify additional variation at the PON gene cluster that improved prediction of PON1 activity and determine if these variants predict carotid artery disease (CAAD). Methods. We considered 1,328 males in a CAAD cohort. 51 tagging single-nucleotide polymorphisms (tag SNPs) across the PON cluster were evaluated to determine their effects on PON1 activity and CAAD status. Results. Six SNPs (four in PON1 and one each in PON2/3) predicted PON1 arylesterase (AREase) activity, in addition to the four previously known functional SNPs. In total, the 10 SNPs explained 30.1% of AREase activity, 5% of which was attributable to the six identified predictive SNPs. We replicate rs854567 prediction of 2.3% of AREase variance, the effects of rs3917510, and a PON3 haplotype that includes rs2375005. While AREase activity strongly predicted CAAD, none of the 10 SNPs predicting AREase predicted CAAD. Conclusions. This study identifies new genetic variants that predict additional PON1 AREase activity. Identification of SNPs associated with PON1 activity is required when evaluating the many phenotypes associated with genetic variation near PON1.

Protectors or Traitors: The Roles of PON2 and PON3 in Atherosclerosis and Cancer

Sun, 13 May 2012 15:37:55 +0000

Cancer and atherosclerosis are major causes of death in western societies. Deregulated cell death is common to both diseases, with significant contribution of inflammatory processes and oxidative stress. These two form a vicious cycle and regulate cell death pathways in either direction. This raises interest in antioxidative systems. The human enzymes paraoxonase-2 (PON2) and PON3 are intracellular enzymes with established antioxidative effects and protective functions against atherosclerosis. Underlying molecular mechanisms, however, remained elusive until recently. Novel findings revealed that both enzymes locate to mitochondrial membranes where they interact with coenzyme Q10 and diminish oxidative stress. As a result, ROS-triggered mitochondrial apoptosis and cell death are reduced. From a cardiovascular standpoint, this is beneficial given that enhanced loss of vascular cells and macrophage death forms the basis for atherosclerotic plaque development. However, the same function has now been shown to raise chemotherapeutic resistance in several cancer cells. Intriguingly, PON2 as well as PON3 are frequently found upregulated in tumor samples. Here we review studies reporting PON2/PON3 deregulations in cancer, summarize most recent findings on their anti-oxidative and antiapoptotic mechanisms, and discuss how this could be used in putative future therapies to target atherosclerosis and cancer.

Interactions between Bile Acids and Nuclear Receptors and Their Effects on Lipid Metabolism and Liver Diseases

Sun, 22 Apr 2012 08:28:58 +0000

A Pleiotropic Role for the Orphan Nuclear Receptor Small Heterodimer Partner in Lipid Homeostasis and Metabolic Pathways

Sun, 22 Apr 2012 08:24:56 +0000

Nuclear receptors (NRs) comprise one of the most abundant classes of transcriptional regulators of metabolic diseases and have emerged as promising pharmaceutical targets. Small heterodimer partner (SHP; NR0B2) is a unique orphan NR lacking a DNA-binding domain but contains a putative ligand-binding domain. SHP is a transcriptional regulator affecting multiple key biological functions and metabolic processes including cholesterol, bile acid, and fatty acid metabolism, as well as reproductive biology and glucose-energy homeostasis. About half of all mammalian NRs and several transcriptional coregulators can interact with SHP. The SHP-mediated repression of target transcription factors includes at least three mechanisms including direct interference with the C-terminal activation function 2 (AF2) coactivator domains of NRs, recruitment of corepressors, or direct interaction with the surface of NR/transcription factors. Future research must focus on synthetic ligands acting on SHP as a potential therapeutic target in a series of metabolic abnormalities. Current understanding about the pleiotropic role of SHP is examined in this paper, and principal metabolic aspects connected with SHP function will be also discussed.

Novel Associations of Nonstructural Loci with Paraoxonase Activity

Tue, 17 Apr 2012 14:30:59 +0000

The high-density-lipoprotein-(HDL-) associated esterase paraoxonase 1 (PON1) is a likely contributor to the antioxidant and antiatherosclerotic capabilities of HDL. Two nonsynonymous mutations in the structural gene, PON1, have been associated with variation in activity levels, but substantial interindividual differences remain unexplained and are greatest for substrates other than the eponymous paraoxon. PON1 activity levels were measured for three substrates—organophosphate paraoxon, arylester phenyl acetate, and lactone dihydrocoumarin—in 767 Mexican American individuals from San Antonio, Texas. Genetic influences on activity levels for each substrate were evaluated by association with approximately one million single nucleotide polymorphism (SNPs) while conditioning on PON1 genotypes. Significant associations were detected at five loci including regions on chromosomes 4 and 17 known to be associated with atherosclerosis and lipoprotein regulation and loci on chromosome 3 that regulate ubiquitous transcription factors. These loci explain 7.8% of variation in PON1 activity with lactone as a substrate, 5.6% with the arylester, and 3.0% with paraoxon. In light of the potential importance of PON1 in preventing cardiovascular disease/events, these novel loci merit further investigation.

Paraoxonases-2 and -3 Are Important Defense Enzymes against Pseudomonas aeruginosa Virulence Factors due to Their Anti-Oxidative and Anti-Inflammatory Properties

Thu, 12 Apr 2012 14:34:57 +0000

The pathogen Pseudomonas aeruginosa causes serious damage in immunocompromised patients by secretion of various virulence factors, among them the quorum sensing N-(3-oxododecanoyl)-L-homoserine lactone (3OC12) and the redox-active pyocyanin (PCN). Paraoxonase-2 (PON2) may protect against P. aeruginosa infections, as it efficiently inactivates 3OC12 and diminishes PCN-induced oxidative stress. This defense could be circumvented because 3OC12 mediates intracellular Ca2+-rise in host cells, which causes rapid inactivation and degradation of PON2. Importantly, we recently found that the PON2 paralogue PON3 prevents mitochondrial radical formation. Here we investigated its role as additional potential defense mechanism against P. aeruginosa infections. Our studies demonstrate that PON3 diminished PCN-induced oxidative stress. Moreover, it showed clear anti-inflammatory potential by protecting against NF-κB activation and IL-8 release. The latter similarly applied to PON2. Furthermore, we observed a Ca2+-mediated inactivation and degradation of PON3, again in accordance with previous findings for PON2. Our results suggest that the anti-oxidative and anti-inflammatory functions of PON2 and PON3 are an important part of our innate defense system against P. aeruginosa infections. Furthermore, we conclude that P. aeruginosa circumvents PON3 protection by the same pathway as for PON2. This may help identifying underlying mechanisms in order to sustain the protection afforded by these enzymes.

Regulation of Hepatic Paraoxonase-1 Expression

Bianca Fuhrman — Tue, 03 Apr 2012 10:38:27 +0000

Serum paraoxonase-1 (PON1) is a member of the paraoxonases family (PON1, PON2, and PON3). PON1 is synthesized and secreted by the liver, and in circulation it is associated with HDL. PON1 has antioxidative properties, which are associated with the enzyme’s capability to decrease oxidative stress in atherosclerotic lesions and to attenuate atherosclerosis development. Epidemiological evidence demonstrates that low PON1 activity is associated with increased risk of cardiovascular events and cardiovascular disease and is an independent risk factor for coronary artery disease. Therefore, pharmacological modulation of PON1 activity or PON1 gene expression could constitute a useful approach for preventing atherosclerosis. A primary determinant of serum PON1 levels is the availability of the enzyme for release by the liver, the principal site of PON1 production. Together with the enzyme secretion rate, enzymatic turnover, and protein stability, the level of PON1 gene expression is a major determinant of PON1 status. This paper summarizes recent progress in understanding the regulation of PON1 expression in hepatocytes.