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1.  Subjects with Low Plasma HDL Cholesterol Levels Are Characterized by an Inflammatory and Oxidative Phenotype 
PLoS ONE  2013;8(11):e78241.
Epidemiological studies have shown that low plasma levels of high-density lipoprotein (HDL) cholesterol are associated with increased risk of cardiovascular disease, but the mechanisms for the possible atheroprotective effects of HDL cholesterol have still not been fully clarified, in particular in relation to clinical studies.
To examine the inflammatory, anti-oxidative and metabolic phenotype of subjects with low plasma HDL cholesterol levels.
Methods and Results
Fifteen subjects with low HDL cholesterol levels (eleven males and four females) and 19 subjects with high HDL (three males and 16 females) were recruited. Low HDL cholesterol was defined as ≤10th age/sex specific percentile and high HDL-C was defined as ≥90 age/sex specific percentile. Inflammatory markers in circulation and PBMC gene expression of cholesterol efflux mediators were measured. Our main findings were: (i) subjects with low plasma HDL cholesterol levels were characterized by increased plasma levels of CRP, MMP-9, neopterin, CXCL16 and ICAM-1 as well as low plasma levels of adiponectin, suggesting an inflammatory phenotype; (ii) these individuals also had reduced paraoxonase (PON)1 activity in plasma and PON2 gene expression in peripheral blood mononuclear cells (PBMC) accompanied by increased plasma levels of oxidized LDL suggesting decreased anti-oxidative capacity; and (iii) PBMC from low HDL subjects also had decreased mRNA levels of ABCA1 and ABCG1, suggesting impaired reverse cholesterol transport.
Subjects with low plasma HDL cholesterol levels are characterized by an inflammatory and oxidative phenotype that could contribute to the increased risk of atherosclerotic disorders in these subjects with low HDL levels.
PMCID: PMC3823918  PMID: 24244297
2.  Lipidomic Profiling of Saccharomyces cerevisiae and Zygosaccharomyces bailii Reveals Critical Changes in Lipid Composition in Response to Acetic Acid Stress 
PLoS ONE  2013;8(9):e73936.
When using microorganisms as cell factories in the production of bio-based fuels or chemicals from lignocellulosic hydrolysate, inhibitory concentrations of acetic acid, released from the biomass, reduce the production rate. The undissociated form of acetic acid enters the cell by passive diffusion across the lipid bilayer, mediating toxic effects inside the cell. In order to elucidate a possible link between lipid composition and acetic acid stress, the present study presents detailed lipidomic profiling of the major lipid species found in the plasma membrane, including glycerophospholipids, sphingolipids and sterols, in Saccharomyces cerevisiae (CEN.PK 113_7D) and Zygosaccharomyces bailii (CBS7555) cultured with acetic acid. Detailed physiological characterization of the response of the two yeasts to acetic acid has also been performed in aerobic batch cultivations using bioreactors. Physiological characterization revealed, as expected, that Z. bailii is more tolerant to acetic acid than S. cerevisiae. Z. bailii grew at acetic acid concentrations above 24 g L−1, while limited growth of S. cerevisiae was observed after 11 h when cultured with only 12 g L−1 acetic acid. Detailed lipidomic profiling using electrospray ionization, multiple-reaction-monitoring mass spectrometry (ESI-MRM-MS) showed remarkable changes in the glycerophospholipid composition of Z. bailii, including an increase in saturated glycerophospholipids and considerable increases in complex sphingolipids in both S. cerevisiae (IPC 6.2×, MIPC 9.1×, M(IP)2C 2.2×) and Z. bailii (IPC 4.9×, MIPC 2.7×, M(IP)2C 2.7×), when cultured with acetic acid. In addition, the basal level of complex sphingolipids was significantly higher in Z. bailii than in S. cerevisiae, further emphasizing the proposed link between lipid saturation, high sphingolipid levels and acetic acid tolerance. The results also suggest that acetic acid tolerance is associated with the ability of a given strain to generate large rearrangements in its lipid profile.
PMCID: PMC3762712  PMID: 24023914
3.  Effect of trans Fatty Acid Intake on LC-MS and NMR Plasma Profiles 
PLoS ONE  2013;8(7):e69589.
The consumption of high levels of industrial trans fatty acids (TFA) has been related to cardiovascular disease, diabetes and sudden cardiac death but the causal mechanisms are not well known. In this study, NMR and LC-MS untargeted metabolomics has been used as an approach to explore the impact of TFA intake on plasma metabolites.
Methodology/Principal Findings
In a double-blinded randomized controlled parallel-group study, 52 overweight postmenopausal women received either partially hydrogenated soybean oil, providing 15.7 g/day of TFA (trans18:1) or control oil with mainly oleic acid for 16 weeks. Subsequent to the intervention period, the subjects participated in a 12-week dietary weight loss program. Before and after the TFA intervention and after the weight loss programme, volunteers participated in an oral glucose tolerance test. PLSDA revealed elevated lipid profiles with TFA intake. NMR indicated up-regulated LDL cholesterol levels and unsaturation. LC-MS profiles demonstrated elevated levels of specific polyunsaturated (PUFA) long-chain phosphatidylcholines (PCs) and a sphingomyelin (SM) which were confirmed with a lipidomics based method. Plasma levels of these markers of TFA intake declined to their low baseline levels after the weight loss program for the TFA group and did not fluctuate for the control group. The marker levels were unaffected by OGTT.
This study demonstrates that intake of TFA affects phospholipid metabolism. The preferential integration of trans18:1 into the sn-1 position of PCs, all containing PUFA in the sn-2 position, could be explained by a general up-regulation in the formation of long-chain PUFAs after TFA intake and/or by specific mobilisation of these fats into PCs. NMR supported these findings by revealing increased unsaturation of plasma lipids in the TFA group. These specific changes in membrane lipid species may be related to the mechanisms of TFA-induced disease but need further validation as risk markers.
Trial registration
Registered at as NCT00655902
PMCID: PMC3726671  PMID: 23922748
4.  Targeted Lipidomics in Drosophila melanogaster Identifies Novel 2-Monoacylglycerols and N-acyl Amides 
PLoS ONE  2013;8(7):e67865.
Lipid metabolism is critical to coordinate organ development and physiology in response to tissue-autonomous signals and environmental cues. Changes to the availability and signaling of lipid mediators can limit competitiveness, adaptation to environmental stressors, and augment pathological processes. Two classes of lipids, the N-acyl amides and the 2-acyl glycerols, have emerged as important signaling molecules in a wide range of species with important signaling properties, though most of what is known about their cellular functions is from mammalian models. Therefore, expanding available knowledge on the repertoire of these lipids in invertebrates will provide additional avenues of research aimed at elucidating biosynthetic, metabolic, and signaling properties of these molecules. Drosophila melanogaster is a commonly used organism to study intercellular communication, including the functions of bioactive lipids. However, limited information is available on the molecular identity of lipids with putative biological activities in Drosophila. Here, we used a targeted lipidomics approach to identify putative signaling lipids in third instar Drosophila larvae, possessing particularly large lipid mass in their fat body. We identified 2-linoleoyl glycerol, 2-oleoyl glycerol, and 45 N-acyl amides in larval tissues, and validated our findings by the comparative analysis of Oregon-RS, Canton-S and w1118 strains. Data here suggest that Drosophila represent another model system to use for the study of 2-acyl glycerol and N-acyl amide signaling.
PMCID: PMC3708943  PMID: 23874457
5.  LipidHome: A Database of Theoretical Lipids Optimized for High Throughput Mass Spectrometry Lipidomics 
PLoS ONE  2013;8(5):e61951.
Protein sequence databases are the pillar upon which modern proteomics is supported, representing a stable reference space of predicted and validated proteins. One example of such resources is UniProt, enriched with both expertly curated and automatic annotations. Taken largely for granted, similar mature resources such as UniProt are not available yet in some other “omics” fields, lipidomics being one of them. While having a seasoned community of wet lab scientists, lipidomics lies significantly behind proteomics in the adoption of data standards and other core bioinformatics concepts. This work aims to reduce the gap by developing an equivalent resource to UniProt called ‘LipidHome’, providing theoretically generated lipid molecules and useful metadata. Using the ‘FASTLipid’ Java library, a database was populated with theoretical lipids, generated from a set of community agreed upon chemical bounds. In parallel, a web application was developed to present the information and provide computational access via a web service. Designed specifically to accommodate high throughput mass spectrometry based approaches, lipids are organised into a hierarchy that reflects the variety in the structural resolution of lipid identifications. Additionally, cross-references to other lipid related resources and papers that cite specific lipids were used to annotate lipid records. The web application encompasses a browser for viewing lipid records and a ‘tools’ section where an MS1 search engine is currently implemented. LipidHome can be accessed at
PMCID: PMC3646891  PMID: 23667450
6.  Mathematical Model of Metabolism and Electrophysiology of Amino Acid and Glucose Stimulated Insulin Secretion: In Vitro Validation Using a β-Cell Line 
PLoS ONE  2013;8(3):e52611.
We integrated biological experimental data with mathematical modelling to gain insights into the role played by L-alanine in amino acid-stimulated insulin secretion (AASIS) and in D-glucose-stimulated insulin secretion (GSIS), details important to the understanding of complex β-cell metabolic coupling relationships. We present an ordinary differential equations (ODEs) based simplified kinetic model of core metabolic processes leading to ATP production (glycolysis, TCA cycle, L-alanine-specific reactions, respiratory chain, ATPase and proton leak) and Ca2+ handling (essential channels and pumps in the plasma membrane) in pancreatic β-cells and relate these to insulin secretion. Experimental work was performed using a clonal rat insulin-secreting cell line (BRIN-BD11) to measure the consumption or production of a range of important biochemical parameters (D-glucose, L-alanine, ATP, insulin secretion) and Ca2+ levels. These measurements were then used to validate the theoretical model and fine-tune the parameters. Mathematical modelling was used to predict L-lactate and L-glutamate concentrations following D-glucose and/or L-alanine challenge and Ca2+ levels upon stimulation with a non metabolizable L-alanine analogue. Experimental data and mathematical model simulations combined suggest that L-alanine produces a potent insulinotropic effect via both a stimulatory impact on β-cell metabolism and as a direct result of the membrane depolarization due to Ca2+ influx triggered by L-alanine/Na+ co-transport. Our simulations indicate that both high intracellular ATP and Ca2+ concentrations are required in order to develop full insulin secretory responses. The model confirmed that K+ATP channel independent mechanisms of stimulation of intracellular Ca2+ levels, via generation of mitochondrial coupling messengers, are essential for promotion of the full and sustained insulin secretion response in β-cells.
PMCID: PMC3592881  PMID: 23520444
7.  Factor Structure Underlying Components of Allostatic Load 
PLoS ONE  2012;7(10):e47246.
Allostatic load is a commonly used metric of health risk based on the hypothesis that recurrent exposure to environmental demands (e.g., stress) engenders a progressive dysregulation of multiple physiological systems. Prominent indicators of response to environmental challenges, such as stress-related hormones, sympatho-vagal balance, or inflammatory cytokines, comprise primary allostatic mediators. Secondary mediators reflect ensuing biological alterations that accumulate over time and confer risk for clinical disease but overlap substantially with a second metric of health risk, the metabolic syndrome. Whether allostatic load mediators covary and thus warrant treatment as a unitary construct remains to be established and, in particular, the relation of allostatic load parameters to the metabolic syndrome requires elucidation. Here, we employ confirmatory factor analysis to test: 1) whether a single common factor underlies variation in physiological systems associated with allostatic load; and 2) whether allostatic load parameters continue to load on a single common factor if a second factor representing the metabolic syndrome is also modeled. Participants were 645 adults from Allegheny County, PA (30–54 years old, 82% non-Hispanic white, 52% female) who were free of confounding medications. Model fitting supported a single, second-order factor underlying variance in the allostatic load components available in this study (metabolic, inflammatory and vagal measures). Further, this common factor reflecting covariation among allostatic load components persisted when a latent factor representing metabolic syndrome facets was conjointly modeled. Overall, this study provides novel evidence that the modeled allostatic load components do share common variance as hypothesized. Moreover, the common variance suggests the existence of statistical coherence above and beyond that attributable to the metabolic syndrome.
PMCID: PMC3480389  PMID: 23112812
8.  Deletion of the metabolic transcriptional coactivator PGC1β induces cardiac arrhythmia 
Cardiovascular Research  2011;92(1):29-38.
Peroxisome proliferator-activated receptor-γ coactivators PGC1α and PGC1β modulate mitochondrial biogenesis and energy homeostasis. The function of these transcriptional coactivators is impaired in obesity, insulin resistance, and type 2 diabetes. We searched for transcriptomic, lipidomic, and electrophysiological alterations in PGC1β−/− hearts potentially associated with increased arrhythmic risk in metabolic diseases.
Methods and results
Microarray analysis in mouse PGC1β−/− hearts confirmed down-regulation of genes related to oxidative phosphorylation and the electron transport chain and up-regulation of hypertrophy- and hypoxia-related genes. Lipidomic analysis showed increased levels of the pro-arrhythmic and pro-inflammatory lipid, lysophosphatidylcholine. PGC1β−/− mouse electrocardiograms showed irregular heartbeats and an increased incidence of polymorphic ventricular tachycardia following isoprenaline infusion. Langendorff-perfused PGC1β−/− hearts showed action potential alternans, early after-depolarizations, and ventricular tachycardia. PGC1β−/− ventricular myocytes showed oscillatory resting potentials, action potentials with early and delayed after-depolarizations, and burst firing during sustained current injection. They showed abnormal diastolic Ca2+ transients, whose amplitude and frequency were increased by isoprenaline, and Ca2+ currents with negatively shifted inactivation characteristics, with increased window currents despite unaltered levels of CACNA1C RNA transcripts. Inwardly and outward rectifying K+ currents were all increased. Quantitiative RT-PCR demonstrated increased SCN5A, KCNA5, RYR2, and Ca2+-calmodulin dependent protein kinase II expression.
PGC1β−/− hearts showed a lysophospholipid-induced cardiac lipotoxicity and impaired bioenergetics accompanied by an ion channel remodelling and altered Ca2+ homeostasis, converging to produce a ventricular arrhythmic phenotype particularly during adrenergic stress. This could contribute to the increased cardiac mortality associated with both metabolic and cardiac disease attributable to lysophospholipid accumulation.
PMCID: PMC3172981  PMID: 21632884
Mitochondria; Cardiac arrhythmia; Peroxisome proliferator-activated receptor-γ coactivator 1β; Metabolic disease; Lysophosphatidylcholine
9.  Metabolomics Reveals Amino Acids Contribute to Variation in Response to Simvastatin Treatment 
PLoS ONE  2012;7(7):e38386.
Statins are widely prescribed for reducing LDL-cholesterol (C) and risk for cardiovascular disease (CVD), but there is considerable variation in therapeutic response. We used a gas chromatography-time-of-flight mass-spectrometry-based metabolomics platform to evaluate global effects of simvastatin on intermediary metabolism. Analyses were conducted in 148 participants in the Cholesterol and Pharmacogenetics study who were profiled pre and six weeks post treatment with 40 mg/day simvastatin: 100 randomly selected from the full range of the LDL-C response distribution and 24 each from the top and bottom 10% of this distribution (“good” and “poor” responders, respectively). The metabolic signature of drug exposure in the full range of responders included essential amino acids, lauric acid (p<0.0055, q<0.055), and alpha-tocopherol (p<0.0003, q<0.017). Using the HumanCyc database and pathway enrichment analysis, we observed that the metabolites of drug exposure were enriched for the pathway class amino acid degradation (p<0.0032). Metabolites whose change correlated with LDL-C lowering response to simvastatin in the full range responders included cystine, urea cycle intermediates, and the dibasic amino acids ornithine, citrulline and lysine. These dibasic amino acids share plasma membrane transporters with arginine, the rate-limiting substrate for nitric oxide synthase (NOS), a critical mediator of cardiovascular health. Baseline metabolic profiles of the good and poor responders were analyzed by orthogonal partial least square discriminant analysis so as to determine the metabolites that best separated the two response groups and could be predictive of LDL-C response. Among these were xanthine, 2-hydroxyvaleric acid, succinic acid, stearic acid, and fructose. Together, the findings from this study indicate that clusters of metabolites involved in multiple pathways not directly connected with cholesterol metabolism may play a role in modulating the response to simvastatin treatment.
Trial Registration NCT00451828
PMCID: PMC3392268  PMID: 22808006
10.  Farnesoid X Receptor Deficiency Improves Glucose Homeostasis in Mouse Models of Obesity 
Diabetes  2011;60(7):1861-1871.
Bile acids (BA) participate in the maintenance of metabolic homeostasis acting through different signaling pathways. The nuclear BA receptor farnesoid X receptor (FXR) regulates pathways in BA, lipid, glucose, and energy metabolism, which become dysregulated in obesity. However, the role of FXR in obesity and associated complications, such as dyslipidemia and insulin resistance, has not been directly assessed.
Here, we evaluate the consequences of FXR deficiency on body weight development, lipid metabolism, and insulin resistance in murine models of genetic and diet-induced obesity.
FXR deficiency attenuated body weight gain and reduced adipose tissue mass in both models. Surprisingly, glucose homeostasis improved as a result of an enhanced glucose clearance and adipose tissue insulin sensitivity. In contrast, hepatic insulin sensitivity did not change, and liver steatosis aggravated as a result of the repression of β-oxidation genes. In agreement, liver-specific FXR deficiency did not protect from diet-induced obesity and insulin resistance, indicating a role for nonhepatic FXR in the control of glucose homeostasis in obesity. Decreasing elevated plasma BA concentrations in obese FXR-deficient mice by administration of the BA sequestrant colesevelam improved glucose homeostasis in a FXR-dependent manner, indicating that the observed improvements by FXR deficiency are not a result of indirect effects of altered BA metabolism.
Overall, FXR deficiency in obesity beneficially affects body weight development and glucose homeostasis.
PMCID: PMC3121443  PMID: 21593203
11.  Farnesoid x receptor deficiency improves glucose homeostasis in mouse models of obesity 
Diabetes  2011;60(7):1861-1871.
Bile acids (BA) participate in the maintenance of metabolic homeostasis acting through different signaling pathways. The nuclear BA receptor farnesoid X receptor (FXR) regulates pathways in BA, lipid, glucose and energy metabolism which become dysregulated in obesity. However, the role of FXR in obesity and associated complications, such as dyslipidemia and insulin resistance, has not been directly assessed.
Research Design and Methods
Here, we evaluate the consequences of FXR-deficiency on body weight development, lipid metabolism and insulin resistance in murine models of genetic and diet-induced obesity.
FXR-deficiency attenuated body weight gain and reduced adipose tissue mass in both models. Surprisingly, glucose homeostasis improved due to an enhanced glucose clearance and adipose tissue insulin sensitivity. In contrast, hepatic insulin sensitivity did not change, and liver steatosis aggravated due to the repression of β-oxidation genes. In agreement, liver-specific FXR-deficiency did not protect from diet-induced obesity and insulin resistance indicating a role for non-hepatic FXR in the control of glucose homeostasis in obesity. Decreasing elevated plasma BA concentrations in obese FXR-deficient mice by administration of the BA sequestrant colesevelam improved glucose homeostasis in a FXR-dependent manner indicating that the observed improvements by FXR-deficiency are not due to indirect effects of altered BA metabolism.
Overall, FXR-deficiency in obesity beneficially affects body weight development and glucose homeostasis.
PMCID: PMC3121443  PMID: 21593203
FXR; bile acids; obesity; glucose homeostasis; insulin resistance; energy metabolism; triglyceride metabolism; bile acid sequestrants
12.  Clustering by Plasma Lipoprotein Profile Reveals Two Distinct Subgroups with Positive Lipid Response to Fenofibrate Therapy 
PLoS ONE  2012;7(6):e38072.
Fibrates lower triglycerides and raise HDL cholesterol in dyslipidemic patients, but show heterogeneous treatment response. We used k-means clustering to identify three representative NMR lipoprotein profiles for 775 subjects from the GOLDN population, and study the response to fenofibrate in corresponding subgroups. The subjects in each subgroup showed differences in conventional lipid characteristics and in presence/absence of cardiovascular risk factors at baseline; there were subgroups with a low, medium and high degree of dyslipidemia. Modeling analysis suggests that the difference between the subgroups with low and medium dyslipidemia is influenced mainly by hepatic uptake dysfunction, while the difference between subgroups with medium and high dyslipidemia is influenced mainly by extrahepatic lipolysis disfunction. The medium and high dyslipidemia subgroups showed a positive, yet distinct lipid response to fenofibrate treatment. When comparing our subgroups to known subgrouping methods, we identified an additional 33% of the population with favorable lipid response to fenofibrate compared to a standard baseline triglyceride cutoff method. Compared to a standard HDL cholesterol cutoff method, the addition was 18%. In conclusion, by using constructing subgroups based on representative lipoprotein profiles, we have identified two subgroups of subjects with positive lipid response to fenofibrate therapy and with different underlying disturbances in lipoprotein metabolism. The total subgroup with positive lipid response to fenofibrate is larger than subgroups identified with baseline triglyceride and HDL cholesterol cutoffs.
PMCID: PMC3373573  PMID: 22719863
13.  Interpretation and Visualization of Non-Linear Data Fusion in Kernel Space: Study on Metabolomic Characterization of Progression of Multiple Sclerosis 
PLoS ONE  2012;7(6):e38163.
In the last decade data fusion has become widespread in the field of metabolomics. Linear data fusion is performed most commonly. However, many data display non-linear parameter dependences. The linear methods are bound to fail in such situations. We used proton Nuclear Magnetic Resonance and Gas Chromatography-Mass Spectrometry, two well established techniques, to generate metabolic profiles of Cerebrospinal fluid of Multiple Sclerosis (MScl) individuals. These datasets represent non-linearly separable groups. Thus, to extract relevant information and to combine them a special framework for data fusion is required.
The main aim is to demonstrate a novel approach for data fusion for classification; the approach is applied to metabolomics datasets coming from patients suffering from MScl at a different stage of the disease. The approach involves data fusion in kernel space and consists of four main steps. The first one is to extract the significant information per data source using Support Vector Machine Recursive Feature Elimination. This method allows one to select a set of relevant variables. In the next step the optimized kernel matrices are merged by linear combination. In step 3 the merged datasets are analyzed with a classification technique, namely Kernel Partial Least Square Discriminant Analysis. In the final step, the variables in kernel space are visualized and their significance established.
We find that fusion in kernel space allows for efficient and reliable discrimination of classes (MScl and early stage). This data fusion approach achieves better class prediction accuracy than analysis of individual datasets and the commonly used mid-level fusion. The prediction accuracy on an independent test set (8 samples) reaches 100%. Additionally, the classification model obtained on fused kernels is simpler in terms of complexity, i.e. just one latent variable was sufficient. Finally, visualization of variables importance in kernel space was achieved.
PMCID: PMC3371049  PMID: 22715376
14.  Efficient and Specific Analysis of Red Blood Cell Glycerophospholipid Fatty Acid Composition 
PLoS ONE  2012;7(3):e33874.
Red blood cell (RBC) n-3 fatty acid status is related to various health outcomes. Accepted biological markers for the fatty acid status determination are RBC phospholipids, phosphatidylcholine, and phosphatidyletholamine. The analysis of these lipid fractions is demanding and time consuming and total phospholipid n-3 fatty acid levels might be affected by changes of sphingomyelin contents in the RBC membrane during n-3 supplementation.
We developed a method for the specific analysis of RBC glycerophospholipids. The application of the new method in a DHA supplementation trial and the comparison to established markers will determine the relevance of RBC GPL as a valid fatty acid status marker in humans.
Methyl esters of glycerophospholipid fatty acids are selectively generated by a two step procedure involving methanolic protein precipitation and base-catalysed methyl ester synthesis. RBC GPL solubilisation is facilitated by ultrasound treatment. Fatty acid status in RBC glycerophospholipids and other established markers were evaluated in thirteen subjects participating in a 30 days supplementation trial (510 mg DHA/d).
The intra-assay CV for GPL fatty acids ranged from 1.0 to 10.5% and the inter-assay CV from 1.3 to 10.9%. Docosahexaenoic acid supplementation significantly increased the docosahexaenoic acid contents in all analysed lipid fractions. High correlations were observed for most of the mono- and polyunsaturated fatty acids, and for the omega-3 index (r = 0.924) between RBC phospholipids and glycerophospholipids. The analysis of RBC glycerophospholipid fatty acids yields faster, easier and less costly results equivalent to the conventional analysis of RBC total phospholipids.
PMCID: PMC3316509  PMID: 22479463
15.  Gender-Divergent Profile of Bile Acid Homeostasis during Aging of Mice 
PLoS ONE  2012;7(3):e32551.
Aging is a physiological process with a progressive decline of adaptation and functional capacity of the body. Bile acids (BAs) have been recognized as signaling molecules regulating the homeostasis of glucose, lipid, and energy. The current study characterizes the age-related changes of individual BA concentrations by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) in serum and liver of male and female C57BL/6 mice from 3 to 27 months of age. Total BA concentrations in serum increased 340% from 3 to 27 months in female mice, whereas they remained relatively constant with age in male mice. During aging, male and female mice shared the following changes: (1) BA concentrations in liver remained relatively constant; (2) the proportions of beta-muricholic acid (βMCA) increased and deoxycholic acid (DCA) decreased between 3 and 27 months in serum and liver; and (3) total BAs in serum and liver became more hydrophilic between 3 and 27 months. In female mice, (1) the mRNAs of hepatic BA uptake transporters, the Na+/taurocholate cotransporting polypeptide (Ntcp) and the organic anion transporting polypeptide 1b2 (Oatp1b2), decreased after 12 months, and similar trends were observed for their proteins; (2) the mRNA of the rate-limiting enzyme for BA synthesis, cholesterol 7α-hydroxylase (Cyp7a1), increased from 3 to 9 months and remained high thereafter. However, in male mice, Ntcp, Oatp1b2, and Cyp7a1 mRNAs remained relatively constant with age. In summary, the current study shows gender-divergent profiles of BA concentrations and composition in serum and liver of mice during aging, which is likely due to the gender-divergent expression of BA transporters Ntcp and Oatp1b2 as well as the synthetic enzyme Cyp7a1.
PMCID: PMC3293819  PMID: 22403674
16.  Potent PPARα Activator Derived from Tomato Juice, 13-oxo-9,11-Octadecadienoic Acid, Decreases Plasma and Hepatic Triglyceride in Obese Diabetic Mice 
PLoS ONE  2012;7(2):e31317.
Dyslipidemia is a major risk factor for development of several obesity-related diseases. The peroxisome proliferator-activated receptor α (PPARα) is a ligand-activated transcription factor that regulates energy metabolism. Previously, we reported that 9-oxo-10,12-octadecadienoic acid (9-oxo-ODA) is presented in fresh tomato fruits and acts as a PPARα agonist. In addition to 9-oxo-ODA, we developed that 13-oxo-9,11-octadecadienoic acid (13-oxo-ODA), which is an isomer of 9-oxo-ODA, is present only in tomato juice. In this study, we explored the possibility that 13-oxo-ODA acts as a PPARα agonist in vitro and whether its effect ameliorates dyslipidemia and hepatic steatosis in vivo. In vitro luciferase assay experiments revealed that 13-oxo-ODA significantly induced PPARα activation; moreover, the luciferase activity of 13-oxo-ODA was stronger than that of 9-oxo-ODA and conjugated linoleic acid (CLA), which is a precursor of 13-oxo-ODA and is well-known as a potent PPARα activator. In addition to in vitro experiment, treatment with 13-oxo-ODA decreased the levels of plasma and hepatic triglycerides in obese KK-Ay mice fed a high-fat diet. In conclusion, our findings indicate that 13-oxo-ODA act as a potent PPARα agonist, suggesting a possibility to improve obesity-induced dyslipidemia and hepatic steatosis.
PMCID: PMC3276502  PMID: 22347463
17.  A “Crossomics” Study Analysing Variability of Different Components in Peripheral Blood of Healthy Caucasoid Individuals 
PLoS ONE  2012;7(1):e28761.
Different immunotherapy approaches for the treatment of cancer and autoimmune diseases are being developed and tested in clinical studies worldwide. Their resulting complex experimental data should be properly evaluated, therefore reliable normal healthy control baseline values are indispensable.
Methodology/Principal Findings
To assess intra- and inter-individual variability of various biomarkers, peripheral blood of 16 age and gender equilibrated healthy volunteers was sampled on 3 different days within a period of one month. Complex “crossomics” analyses of plasma metabolite profiles, antibody concentrations and lymphocyte subset counts as well as whole genome expression profiling in CD4+T and NK cells were performed. Some of the observed age, gender and BMI dependences are in agreement with the existing knowledge, like negative correlation between sex hormone levels and age or BMI related increase in lipids and soluble sugars. Thus we can assume that the distribution of all 39.743 analysed markers is well representing the normal Caucasoid population. All lymphocyte subsets, 20% of metabolites and less than 10% of genes, were identified as highly variable in our dataset.
Our study shows that the intra-individual variability was at least two-fold lower compared to the inter-individual one at all investigated levels, showing the importance of personalised medicine approach from yet another perspective.
PMCID: PMC3257221  PMID: 22253695
18.  Elongase Reactions as Control Points in Long-Chain Polyunsaturated Fatty Acid Synthesis 
PLoS ONE  2011;6(12):e29662.
Δ6-Desaturase (Fads2) is widely regarded as rate-limiting in the conversion of dietary α-linolenic acid (18:3n-3; ALA) to the long-chain omega-3 polyunsaturated fatty acid docosahexaenoic acid (22:6n-3; DHA). However, increasing dietary ALA or the direct Fads2 product, stearidonic acid (18:4n-3; SDA), increases tissue levels of eicosapentaenoic acid (20:5n-3; EPA) and docosapentaenoic acid (22:5n-3; DPA), but not DHA. These observations suggest that one or more control points must exist beyond ALA metabolism by Fads2. One possible control point is a second reaction involving Fads2 itself, since this enzyme catalyses desaturation of 24:5n-3 to 24:6n-3, as well as ALA to SDA. However, metabolism of EPA and DPA both require elongation reactions. This study examined the activities of two elongase enzymes as well as the second reaction of Fads2 in order to concentrate on the metabolism of EPA to DHA.
Methodology/Principal Findings
The substrate selectivities, competitive substrate interactions and dose response curves of the rat elongases, Elovl2 and Elovl5 were determined after expression of the enzymes in yeast. The competitive substrate interactions for rat Fads2 were also examined. Rat Elovl2 was active with C20 and C22 polyunsaturated fatty acids and this single enzyme catalysed the sequential elongation reactions of EPA→DPA→24:5n-3. The second reaction DPA→24:5n-3 appeared to be saturated at substrate concentrations not saturating for the first reaction EPA→DPA. ALA dose-dependently inhibited Fads2 conversion of 24:5n-3 to 24:6n-3.
The competition between ALA and 24:5n-3 for Fads2 may explain the decrease in DHA levels observed after certain intakes of dietary ALA have been exceeded. In addition, the apparent saturation of the second Elovl2 reaction, DPA→24:5n-3, provides further explanations for the accumulation of DPA when ALA, SDA or EPA is provided in the diet. This study suggests that Elovl2 will be critical in understanding if DHA synthesis can be increased by dietary means.
PMCID: PMC3245304  PMID: 22216341
19.  T Cells Recognizing a Peptide Contaminant Undetectable by Mass Spectrometry 
PLoS ONE  2011;6(12):e28866.
Synthetic peptides are widely used in immunological research as epitopes to stimulate their cognate T cells. These preparations are never completely pure, but trace contaminants are commonly revealed by mass spectrometry quality controls. In an effort to characterize novel major histocompatibility complex (MHC) Class I-restricted β-cell epitopes in non-obese diabetic (NOD) mice, we identified islet-infiltrating CD8+ T cells recognizing a contaminating peptide. The amount of this contaminant was so small to be undetectable by direct mass spectrometry. Only after concentration by liquid chromatography, we observed a mass peak corresponding to an immunodominant islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)206-214 epitope described in the literature. Generation of CD8+ T-cell clones recognizing IGRP206-214 using a novel method confirmed the identity of the contaminant, further underlining the immunodominance of IGRP206-214. If left undetected, minute impurities in synthetic peptide preparations may thus give spurious results.
PMCID: PMC3237501  PMID: 22194932
20.  Alkyl-Glycerol Rescues Plasmalogen Levels and Pathology of Ether-Phospholipid Deficient Mice 
PLoS ONE  2011;6(12):e28539.
A deficiency of plasmalogens, caused by impaired peroxisomal metabolism affects normal development and multiple organs in adulthood. Treatment options aimed at restoring plasmalogen levels may be relevant for the therapy of peroxisomal and non-peroxisomal disorders. In this study we determined the in vivo efficacy of an alkyl glycerol (AG), namely, 1-O-octadecyl-rac-glycerol, as a therapeutic agent for defects in plasmalogen synthesis. To achieve this, Pex7 knockout mice, a mouse model for Rhizomelic Chondrodysplasia Punctata type 1 characterized by the absence of plasmalogens, and WT mice were fed a control diet or a diet containing 2% alkyl-glycerol. Plasmalogen levels were measured in target organs and the biochemical data were correlated with the histological analysis of affected organs. Plasmalogen levels in all peripheral tissues of Pex7 KO mice fed the AG diet for 2 months normalized to the levels of AG fed WT mice. In nervous tissues of Pex7 KO mice fed the AG-diet, plasmalogen levels were significantly increased compared to control fed KO mice. Histological analysis of target organs revealed that the AG-diet was able to stop the progression of the pathology in testis, adipose tissue and the Harderian gland. Interestingly, the latter tissues are characterized by the presence of lipid droplets which were absent or reduced in size and number when ether-phospholipids are lacking, but which can be restored with the AAG treatment. Furthermore, nerve conduction in peripheral nerves was improved. When given prior to the occurrence of major pathological changes, the AG-diet prevented or ameliorated the pathology observed in Pex7 KO mice depending on the degree of plasmalogen restoration. This study provides evidence of the beneficial effects of treating a plasmalogen deficiency with alkyl-glycerol.
PMCID: PMC3232224  PMID: 22163031
21.  Microsecond Molecular Dynamics Simulations of Intrinsically Disordered Proteins Involved in the Oxidative Stress Response 
PLoS ONE  2011;6(11):e27371.
Intrinsically disordered proteins (IDPs) are abundant in cells and have central roles in protein-protein interaction networks. Interactions between the IDP Prothymosin alpha (ProTα) and the Neh2 domain of Nuclear factor erythroid 2-related factor 2 (Nrf2), with a common binding partner, Kelch-like ECH-associated protein 1(Keap1), are essential for regulating cellular response to oxidative stress. Misregulation of this pathway can lead to neurodegenerative diseases, premature aging and cancer. In order to understand the mechanisms these two disordered proteins employ to bind to Keap1, we performed extensive 0.5–1.0 microsecond atomistic molecular dynamics (MD) simulations and isothermal titration calorimetry experiments to investigate the structure/dynamics of free-state ProTα and Neh2 and their thermodynamics of bindings. The results show that in their free states, both ProTα and Neh2 have propensities to form bound-state-like β-turn structures but to different extents. We also found that, for both proteins, residues outside the Keap1-binding motifs may play important roles in stabilizing the bound-state-like structures. Based on our findings, we propose that the binding of disordered ProTα and Neh2 to Keap1 occurs synergistically via preformed structural elements (PSEs) and coupled folding and binding, with a heavy bias towards PSEs, particularly for Neh2. Our results provide insights into the molecular mechanisms Neh2 and ProTα bind to Keap1, information that is useful for developing therapeutics to enhance the oxidative stress response.
PMCID: PMC3220680  PMID: 22125611
22.  Bile Acid Metabolites in Serum: Intraindividual Variation and Associations with Coronary Heart Disease, Metabolic Syndrome and Diabetes Mellitus 
PLoS ONE  2011;6(11):e25006.
Bile acids (BAs) regulate glucose and lipid metabolism. In longitudinal and case-control-studies, we investigated the diurnal variation of serum concentrations of the 15 major BAs as well as the biosynthetic precursor 7α-hydroxy-4-cholesten-3-one (C4) and their associations, respectively, with coronary artery disease (CAD), diabetes mellitus type 2 (T2DM), and non-diabetic metabolic syndrome (MetS). In hourly taken blood samples of four healthy probands, the intraindividual 24 h variation of C4, conjugated and unconjugated BAs ranged from 42% to 72%, from 23% to 91%, and from 49% to 90%, respectively. Conjugated BA concentrations mainly increased following food intake. Serum levels of C4 and unconjugated BAs changed with daytime with maxima varying interindividually between 20h00 and 1h00 and between 3h00 and 8h00, respectively. Comparisons of data from 75 CAD patients with 75 CAD-free controls revealed no statistically significant association of CAD with BAs or C4. Comparisons of data from 50 controls free of T2DM or MetS, 50 MetS patients, and 50 T2DM patients revealed significantly increased fasting serum levels of C4 in patients with MetS and T2DM. Multiple regression analysis revealed body mass index (BMI) and plasma levels of triglycerides (TG) as independent determinants of C4 levels. Upon multivariate and principle component analyses the association of C4 with T2DM and/or MetS was not independent of or superior to the canonical MetS components. In conclusion, despite large intra- and interindividual variation, serum levels of C4,are significantly increased in patients with MetS and T2DM but confounded with BMI and TG.
PMCID: PMC3215718  PMID: 22110577
23.  Nicotinamide Phosphoribosyltransferase/Visfatin Does Not Catalyze Nicotinamide Mononucleotide Formation in Blood Plasma 
PLoS ONE  2011;6(8):e22781.
Nicotinamide (Nam) phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in mammalian NAD synthesis, catalyzing nicotinamide mononucleotide (NMN) formation from Nam and 5-phosphoribosyl 1-pyrophosphate (PRPP). NAMPT has also been described as an adipocytokine visfatin with a variety of actions, although physiological significance of this protein remains unclear. It has been proposed that possible actions of visfatin are mediated through the extracellular formation of NMN. However, we did not detect NMN in mouse blood plasma, even with a highly specific and sensitive liquid chromatography/tandem mass spectrometry. Furthermore, there is no or little ATP, the activator of NAMPT, in extracellular spaces. We thus questioned whether visfatin catalyzes the in situ formation of NMN under such extracellular milieus. To address this question, we here determined Km values for the substrates Nam and PRPP in the NAMPT reaction without or with ATP using a recombinant human enzyme and found that 1 mM ATP dramatically decreases Km values for the substrates, in particular PRPP to its intracellular concentration. Consistent with the kinetic data, only when ATP is present at millimolar levels, NAMPT efficiently catalyzed the NMN formation at the intracellular concentrations of the substrates. Much lower concentrations of Nam and almost the absence of PRPP and ATP in the blood plasma suggest that NAMPT should not efficiently catalyze its reaction under the extracellular milieu. Indeed, NAMPT did not form NMN in the blood plasma. From these kinetic analyses of the enzyme and quantitative determination of its substrates, activator, and product, we conclude that visfatin does not participate in NMN formation under the extracellular milieus. Together with the absence of NMN in the blood plasma, our conclusion does not support the concept of “NAMPT-mediated systemic NAD biosynthesis.” Our study would advance current understanding of visfatin physiology.
PMCID: PMC3149623  PMID: 21826208
24.  Information Dynamics in Living Systems: Prokaryotes, Eukaryotes, and Cancer 
PLoS ONE  2011;6(7):e22085.
Living systems use information and energy to maintain stable entropy while far from thermodynamic equilibrium. The underlying first principles have not been established.
We propose that stable entropy in living systems, in the absence of thermodynamic equilibrium, requires an information extremum (maximum or minimum), which is invariant to first order perturbations. Proliferation and death represent key feedback mechanisms that promote stability even in a non-equilibrium state. A system moves to low or high information depending on its energy status, as the benefit of information in maintaining and increasing order is balanced against its energy cost. Prokaryotes, which lack specialized energy-producing organelles (mitochondria), are energy-limited and constrained to an information minimum. Acquisition of mitochondria is viewed as a critical evolutionary step that, by allowing eukaryotes to achieve a sufficiently high energy state, permitted a phase transition to an information maximum. This state, in contrast to the prokaryote minima, allowed evolution of complex, multicellular organisms. A special case is a malignant cell, which is modeled as a phase transition from a maximum to minimum information state. The minimum leads to a predicted power-law governing the in situ growth that is confirmed by studies measuring growth of small breast cancers.
We find living systems achieve a stable entropic state by maintaining an extreme level of information. The evolutionary divergence of prokaryotes and eukaryotes resulted from acquisition of specialized energy organelles that allowed transition from information minima to maxima, respectively. Carcinogenesis represents a reverse transition: of an information maximum to minimum. The progressive information loss is evident in accumulating mutations, disordered morphology, and functional decline characteristics of human cancers. The findings suggest energy restriction is a critical first step that triggers the genetic mutations that drive somatic evolution of the malignant phenotype.
PMCID: PMC3139603  PMID: 21818295
25.  Bioinformatic and Genetic Association Analysis of MicroRNA Target Sites in One-Carbon Metabolism Genes 
PLoS ONE  2011;6(7):e21851.
One-carbon metabolism (OCM) is linked to DNA synthesis and methylation, amino acid metabolism and cell proliferation. OCM dysfunction has been associated with increased risk for various diseases, including cancer and neural tube defects. MicroRNAs (miRNAs) are ∼22 nt RNA regulators that have been implicated in a wide array of basic cellular processes, such as differentiation and metabolism. Accordingly, mis-regulation of miRNA expression and/or activity can underlie complex disease etiology. We examined the possibility of OCM regulation by miRNAs. Using computational miRNA target prediction methods and Monte-Carlo based statistical analyses, we identified two candidate miRNA “master regulators” (miR-22 and miR-125) and one candidate pair of “master co-regulators” (miR-344-5p/484 and miR-488) that may influence the expression of a significant number of genes involved in OCM. Interestingly, miR-22 and miR-125 are significantly up-regulated in cells grown under low-folate conditions. In a complementary analysis, we identified 15 single nucleotide polymorphisms (SNPs) that are located within predicted miRNA target sites in OCM genes. We genotyped these 15 SNPs in a population of healthy individuals (age 18–28, n = 2,506) that was previously phenotyped for various serum metabolites related to OCM. Prior to correction for multiple testing, we detected significant associations between TCblR rs9426 and methylmalonic acid (p  =  0.045), total homocysteine levels (tHcy) (p  =  0.033), serum B12 (p < 0.0001), holo transcobalamin (p < 0.0001) and total transcobalamin (p < 0.0001); and between MTHFR rs1537514 and red blood cell folate (p < 0.0001). However, upon further genetic analysis, we determined that in each case, a linked missense SNP is the more likely causative variant. Nonetheless, our Monte-Carlo based in silico simulations suggest that miRNAs could play an important role in the regulation of OCM.
PMCID: PMC3134459  PMID: 21765920

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