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1.
J Proteome Res ; 23(8): 3496-3514, 2024 Aug 02.
Article in English | MEDLINE | ID: mdl-38986055

ABSTRACT

Dupilumab is a monoclonal antibody approved for the treatment of atopic dermatitis (AD); however, its effects on molecular, cellular, and immunological levels remain to be elucidated. In this study, blood and dermal interstitial fluid (ISF) from nonlesional (NL) and lesional (L) skin were collected from eight patients with moderate to severe AD, before (visit 2-v2) and at the end of a 16-week treatment with dupilumab (visit 10-v10). Clinical treatment effect was demonstrated by significantly decreased AD severity scores at the end of treatment. At v10 versus v2, the percentages of CD4+ interleukin-producing cells showed a decreasing trend in ISF L and NL, unbound IL-4 levels in plasma were increased, IL-5 levels in ISF L reduced, and levels of factors involved in anti-inflammatory pathways and re-epithelization increased. At v2, ISF L showed that AD lesions might have altered amino acid pathways and lipid signaling compared to ISF NL. At v10, ISF L exhibited raised levels of long- and very-long-chain fatty acids and lipids compared to v2. Furthermore, dupilumab administration caused reduced expression of miR-155-5p and miR-378a-3p in ISF L. In conclusion, results from the present study provided novel knowledge by linking local immune and metabolic alterations to AD pathogenesis and treatment response.


Subject(s)
Antibodies, Monoclonal, Humanized , Dermatitis, Atopic , Extracellular Fluid , Humans , Dermatitis, Atopic/drug therapy , Dermatitis, Atopic/metabolism , Antibodies, Monoclonal, Humanized/pharmacology , Antibodies, Monoclonal, Humanized/therapeutic use , Extracellular Fluid/drug effects , Extracellular Fluid/metabolism , Male , Female , Adult , Inflammation Mediators/metabolism , Metabolome/drug effects , Interleukin-4/metabolism , MicroRNAs/metabolism , MicroRNAs/genetics , Skin/metabolism , Skin/drug effects , Skin/blood supply , Middle Aged , Interleukin-5
2.
Drug Resist Updat ; 71: 100993, 2023 Nov.
Article in English | MEDLINE | ID: mdl-37639774

ABSTRACT

AIMS: Drivers of the drug tolerant proliferative persister (DTPP) state have not been well investigated. Histone H3 lysine-4 trimethylation (H3K4me3), an active histone mark, might enable slow cycling drug tolerant persisters (DTP) to regain proliferative capacity. This study aimed to determine H3K4me3 transcriptionally active sites identifying a key regulator of DTPPs. METHODS: Deploying a model of adaptive cancer drug tolerance, H3K4me3 ChIP-Seq data of DTPPs guided identification of top transcription factor binding motifs. These suggested involvement of O-linked N-acetylglucosamine transferase (OGT), which was confirmed by metabolomics analysis and biochemical assays. OGT impact on DTPPs and adaptive resistance was explored in vitro and in vivo. RESULTS: H3K4me3 remodeling was widespread in CPG island regions and DNA binding motifs associated with O-GlcNAc marked chromatin. Accordingly, we observed an upregulation of OGT, O-GlcNAc and its binding partner TET1 in chronically treated cancer cells. Inhibition of OGT led to loss of H3K4me3 and downregulation of genes contributing to drug resistance. Genetic ablation of OGT prevented acquired drug resistance in in vivo models. Upstream of OGT, we identified AMPK as an actionable target. AMPK activation by acetyl salicylic acid downregulated OGT with similar effects on delaying acquired resistance. CONCLUSION: Our findings uncover a fundamental mechanism of adaptive drug resistance that governs cancer cell reprogramming towards acquired drug resistance, a process that can be exploited to improve response duration and patient outcomes.


Subject(s)
AMP-Activated Protein Kinases , Histones , Humans , Histones/genetics , Down-Regulation , Mixed Function Oxygenases , Proto-Oncogene Proteins
3.
Basic Res Cardiol ; 117(1): 45, 2022 09 06.
Article in English | MEDLINE | ID: mdl-36068416

ABSTRACT

Tachycardiomyopathy is characterised by reversible left ventricular dysfunction, provoked by rapid ventricular rate. While the knowledge of mitochondria advanced in most cardiomyopathies, mitochondrial functions await elucidation in tachycardiomyopathy. Pacemakers were implanted in 61 rabbits. Tachypacing was performed with 330 bpm for 10 days (n = 11, early left ventricular dysfunction) or with up to 380 bpm over 30 days (n = 24, tachycardiomyopathy, TCM). In n = 26, pacemakers remained inactive (SHAM). Left ventricular tissue was subjected to respirometry, metabolomics and acetylomics. Results were assessed for translational relevance using a human-based model: induced pluripotent stem cell derived cardiomyocytes underwent field stimulation for 7 days (TACH-iPSC-CM). TCM animals showed systolic dysfunction compared to SHAM (fractional shortening 37.8 ± 1.0% vs. 21.9 ± 1.2%, SHAM vs. TCM, p < 0.0001). Histology revealed cardiomyocyte hypertrophy (cross-sectional area 393.2 ± 14.5 µm2 vs. 538.9 ± 23.8 µm2, p < 0.001) without fibrosis. Mitochondria were shifted to the intercalated discs and enlarged. Mitochondrial membrane potential remained stable in TCM. The metabolite profiles of ELVD and TCM were characterised by profound depletion of tricarboxylic acid cycle intermediates. Redox balance was shifted towards a more oxidised state (ratio of reduced to oxidised nicotinamide adenine dinucleotide 10.5 ± 2.1 vs. 4.0 ± 0.8, p < 0.01). The mitochondrial acetylome remained largely unchanged. Neither TCM nor TACH-iPSC-CM showed relevantly increased levels of reactive oxygen species. Oxidative phosphorylation capacity of TCM decreased modestly in skinned fibres (168.9 ± 11.2 vs. 124.6 ± 11.45 pmol·O2·s-1·mg-1 tissue, p < 0.05), but it did not in isolated mitochondria. The pattern of mitochondrial dysfunctions detected in two models of tachycardiomyopathy diverges from previously published characteristic signs of other heart failure aetiologies.


Subject(s)
Cardiomyopathies , Heart Failure , Ventricular Dysfunction, Left , Animals , Cardiomyopathies/etiology , Humans , Mitochondria/metabolism , Myocardium/metabolism , Rabbits
4.
Mol Cell ; 53(5): 710-25, 2014 Mar 06.
Article in English | MEDLINE | ID: mdl-24560926

ABSTRACT

Acetyl-coenzyme A (AcCoA) is a major integrator of the nutritional status at the crossroads of fat, sugar, and protein catabolism. Here we show that nutrient starvation causes rapid depletion of AcCoA. AcCoA depletion entailed the commensurate reduction in the overall acetylation of cytoplasmic proteins, as well as the induction of autophagy, a homeostatic process of self-digestion. Multiple distinct manipulations designed to increase or reduce cytosolic AcCoA led to the suppression or induction of autophagy, respectively, both in cultured human cells and in mice. Moreover, maintenance of high AcCoA levels inhibited maladaptive autophagy in a model of cardiac pressure overload. Depletion of AcCoA reduced the activity of the acetyltransferase EP300, and EP300 was required for the suppression of autophagy by high AcCoA levels. Altogether, our results indicate that cytosolic AcCoA functions as a central metabolic regulator of autophagy, thus delineating AcCoA-centered pharmacological strategies that allow for the therapeutic manipulation of autophagy.


Subject(s)
Acetyl Coenzyme A/chemistry , Autophagy , Cytosol/enzymology , Gene Expression Regulation, Enzymologic , Adenosine Triphosphate/chemistry , Animals , Cell Line, Tumor , Cell Nucleus/metabolism , Cytoplasm/metabolism , Cytosol/metabolism , E1A-Associated p300 Protein/chemistry , Green Fluorescent Proteins/metabolism , HCT116 Cells , HeLa Cells , Humans , Ketoglutaric Acids/chemistry , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence , Mitochondria/metabolism , RNA, Small Interfering/metabolism
5.
Int J Mol Sci ; 23(14)2022 Jul 19.
Article in English | MEDLINE | ID: mdl-35887308

ABSTRACT

(1) The cardio-reno-metabolic benefits of the SGLT2 inhibitors canagliflozin (cana), dapagliflozin (dapa), ertugliflozin (ertu), and empagliflozin (empa) have been demonstrated, but it remains unclear whether they exert different off-target effects influencing clinical profiles. (2) We aimed to investigate the effects of SGLT2 inhibitors on mitochondrial function, cellular glucose-uptake (GU), and metabolic pathways in human-umbilical-vein endothelial cells (HUVECs). (3) At 100 µM (supra-pharmacological concentration), cana decreased ECAR by 45% and inhibited GU (IC5o: 14 µM). At 100 µM and 10 µM (pharmacological concentration), cana increased the ADP/ATP ratio, whereas dapa and ertu (3, 10 µM, about 10× the pharmacological concentration) showed no effect. Cana (100 µM) decreased the oxygen consumption rate (OCR) by 60%, while dapa decreased it by 7%, and ertu and empa (all 100 µM) had no significant effect. Cana (100 µM) inhibited GLUT1, but did not significantly affect GLUTs' expression levels. Cana (100 µM) treatment reduced glycolysis, elevated the amino acids supplying the tricarboxylic-acid cycle, and significantly increased purine/pyrimidine-pathway metabolites, in contrast to dapa (3 µM) and ertu (10 µM). (4) The results confirmed cana´s inhibition of mitochondrial activity and GU at supra-pharmacological and pharmacological concentrations, whereas the dapa, ertu, and empa did not show effects even at supra-pharmacological concentrations. At supra-pharmacological concentrations, cana (but not dapa or ertu) affected multiple cellular pathways and inhibited GLUT1.


Subject(s)
Diabetes Mellitus, Type 2 , Sodium-Glucose Transporter 2 Inhibitors , Benzhydryl Compounds/pharmacology , Canagliflozin/pharmacology , Diabetes Mellitus, Type 2/drug therapy , Endothelial Cells , Glucose , Glucose Transporter Type 1 , Humans , Mitochondria , Oxidative Phosphorylation , Sodium-Glucose Transporter 2 Inhibitors/pharmacology
6.
Int J Mol Sci ; 23(22)2022 Nov 15.
Article in English | MEDLINE | ID: mdl-36430566

ABSTRACT

Coronavirus disease 2019 (COVID-19)-induced metabolic alterations have been proposed as a source for prognostic biomarkers and may harbor potential for therapeutic exploitation. However, the metabolic impact of COVID-19 in hemodialysis (HD), a setting of profound a priori alterations, remains unstudied. To evaluate potential COVID-19 biomarkers in end-stage kidney disease (CKD G5), we analyzed the plasma metabolites in different COVID-19 stages in patients with or without HD. We recruited 18 and 9 asymptomatic and mild, 11 and 11 moderate, 2 and 13 severely affected, and 10 and 6 uninfected HD and non-HD patients, respectively. Plasma samples were taken at the time of diagnosis and/or upon admission to the hospital and analyzed by targeted metabolomics and cytokine/chemokine profiling. Targeted metabolomics confirmed stage-dependent alterations of the metabolome in non-HD patients with COVID-19, which were less pronounced in HD patients. Elevated kynurenine levels and lipid dysregulation, shown by an increase in circulating free fatty acids and a decrease in lysophospholipids, could distinguish patients with moderate COVID-19 from non-infected individuals in both groups. Kynurenine and lipid alterations were also associated with ICAM-1 and IL-15 levels in HD and non-HD patients. Our findings support the kynurenine pathway and plasma lipids as universal biomarkers of moderate and severe COVID-19 independent of kidney function.


Subject(s)
COVID-19 , Kynurenine , Humans , Tryptophan , Renal Dialysis , Lipids
7.
Am J Physiol Lung Cell Mol Physiol ; 319(2): L312-L324, 2020 08 01.
Article in English | MEDLINE | ID: mdl-32521164

ABSTRACT

Obesity is associated with lung function impairment and respiratory diseases; however, the underlying pathophysiological mechanisms are still elusive, and therapeutic options are limited. This study examined the effects of prolonged excess fat intake on lung mechanics and microstructure and tested spermidine supplementation and physical activity as intervention strategies. C57BL/6N mice fed control diet (10% fat) or high-fat diet (HFD; 60% fat) were left untreated or were supplemented with 3 mM spermidine, had access to running wheels for voluntary activity, or a combination of both. After 30 wk, lung mechanics was assessed, and left lungs were analyzed by design-based stereology. HFD exerted minor effects on lung mechanics and resulted in higher body weight and elevated lung, air, and septal volumes. The number of alveoli was higher in HFD-fed animals. This was accompanied by an increase in epithelial, but not endothelial, surface area. Moreover, air-blood barrier and endothelium were significantly thicker. Neither treatment affected HFD-related body weights. Spermidine lowered lung volumes as well as endothelial and air-blood barrier thicknesses toward control levels and substantially increased the endothelial surface area under HFD. Activity resulted in decreased volumes of lung, septa, and septal compartments but did not affect vascular changes in HFD-fed mice. The combination treatment showed no additive effect. In conclusion, excess fat consumption induced alveolar capillary remodeling indicative of impaired perfusion and gas diffusion. Spermidine alleviated obesity-related endothelial alterations, indicating a beneficial effect, whereas physical activity reduced lung volumes apparently by other, possibly systemic effects.


Subject(s)
Lung/drug effects , Obesity/complications , Obesity/physiopathology , Spermidine/administration & dosage , Animal Feed , Animals , Body Weight/drug effects , Diet, High-Fat/adverse effects , Dietary Supplements , Male , Mice , Mice, Inbred C57BL , Weight Gain/drug effects
8.
FASEB J ; 33(12): 13808-13824, 2019 12.
Article in English | MEDLINE | ID: mdl-31638418

ABSTRACT

N-acetylaspartate (NAA) is synthesized by aspartate N-acetyltransferase (gene: Nat8l) from acetyl-coenzyme A and aspartate. In the brain, NAA is considered an important energy metabolite for lipid synthesis. However, the role of NAA in peripheral tissues remained elusive. Therefore, we characterized the metabolic phenotype of knockout (ko) and adipose tissue-specific (ako) Nat8l-ko mice as well as NAA-supplemented mice on various diets. We identified an important role of NAA availability in the brain during adolescence, as 75% of Nat8l-ko mice died on fat-free diet (FFD) after weaning but could be rescued by NAA supplementation. In adult life, NAA deficiency promotes a beneficial metabolic phenotype, as Nat8l-ko and Nat8l-ako mice showed reduced body weight, increased energy expenditure, and improved glucose tolerance on chow, high-fat, and FFDs. Furthermore, Nat8l-deficient adipocytes exhibited increased mitochondrial respiration, ATP synthesis, and an induction of browning. Conversely, NAA-treated wild-type mice showed reduced adipocyte respiration and lipolysis and increased de novo lipogenesis, culminating in reduced energy expenditure, glucose tolerance, and insulin sensitivity. Mechanistically, our data point to a possible role of NAA as modulator of pancreatic insulin secretion and suggest NAA as a critical energy metabolite for adipocyte and whole-body energy homeostasis.-Hofer, D. C., Zirkovits, G., Pelzmann, H. J., Huber, K., Pessentheiner, A. R., Xia, W., Uno, K., Miyazaki, T., Kon, K., Tsuneki, H., Pendl, T., Al Zoughbi, W., Madreiter-Sokolowski, C. T., Trausinger, G., Abdellatif, M., Schoiswohl, G., Schreiber, R., Eisenberg, T., Magnes, C., Sedej, S., Eckhardt, M., Sasahara, M., Sasaoka, T., Nitta, A., Hoefler, G., Graier, W. F., Kratky, D., Auwerx, J., Bogner-Strauss, J. G. N-acetylaspartate availability is essential for juvenile survival on fat-free diet and determines metabolic health.


Subject(s)
Aspartic Acid/analogs & derivatives , Acetyl Coenzyme A/metabolism , Acetyltransferases/metabolism , Adipocytes/metabolism , Animals , Aspartic Acid/metabolism , Brain/metabolism , Diet, Fat-Restricted , Energy Metabolism/physiology , Insulin Resistance/physiology , Lipolysis/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitochondria/metabolism
9.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1863(4): 467-478, 2018 Apr.
Article in English | MEDLINE | ID: mdl-29374543

ABSTRACT

Lysosomal acid lipase (LAL) is the only known enzyme, which hydrolyzes cholesteryl esters and triacylglycerols in lysosomes of multiple cells and tissues. Here, we explored the role of LAL in brown adipose tissue (BAT). LAL-deficient (Lal-/-) mice exhibit markedly reduced UCP1 expression in BAT, modified BAT morphology with accumulation of lysosomes, and mitochondrial dysfunction, consequently leading to regular hypothermic events in mice kept at room temperature. Cold exposure resulted in reduced lipid uptake into BAT, thereby aggravating dyslipidemia and causing life threatening hypothermia in Lal-/- mice. Linking LAL as a potential regulator of lipoprotein lipase activity, we found Angptl4 mRNA expression upregulated in BAT. Our data demonstrate that LAL is critical for shuttling fatty acids derived from circulating lipoproteins to BAT during cold exposure. We conclude that inhibited lysosomal lipid hydrolysis in BAT leads to impaired thermogenesis in Lal-/- mice.


Subject(s)
Adipose Tissue, Brown/metabolism , Fatty Acids/metabolism , Sterol Esterase/metabolism , Thermogenesis , Acetyl Coenzyme A/metabolism , Adipocytes, Brown/metabolism , Adipose Tissue, Brown/ultrastructure , Animals , Autophagy , Body Temperature , Carnitine/analogs & derivatives , Carnitine/metabolism , Cold Temperature , Disease Progression , Dyslipidemias/metabolism , Dyslipidemias/pathology , Energy Metabolism , Glucose/metabolism , Hypothermia, Induced , Lipid Droplets/metabolism , Lipolysis , Male , Mice, Inbred C57BL , Muscles/metabolism , Oxidation-Reduction , Oxygen Consumption , Sterol Esterase/deficiency , Uncoupling Protein 1/metabolism
10.
Diabetologia ; 59(8): 1743-52, 2016 08.
Article in English | MEDLINE | ID: mdl-27153842

ABSTRACT

AIMS/HYPOTHESIS: Lysosomal acid lipase (LAL) hydrolyses cholesteryl esters and triacylglycerols (TG) within lysosomes to mobilise NEFA and cholesterol. Since LAL-deficient (Lal (-/-) ) mice suffer from progressive loss of adipose tissue and severe accumulation of lipids in hepatic lysosomes, we hypothesised that LAL deficiency triggers alternative energy pathway(s). METHODS: We studied metabolic adaptations in Lal (-/-) mice. RESULTS: Despite loss of adipose tissue, Lal (-/-) mice show enhanced glucose clearance during insulin and glucose tolerance tests and have increased uptake of [(3)H]2-deoxy-D-glucose into skeletal muscle compared with wild-type mice. In agreement, fasted Lal (-/-) mice exhibit reduced glucose and glycogen levels in skeletal muscle. We observed 84% decreased plasma leptin levels and significantly reduced hepatic ATP, glucose, glycogen and glutamine concentrations in fed Lal (-/-) mice. Markedly reduced hepatic acyl-CoA concentrations decrease the expression of peroxisome proliferator-activated receptor α (PPARα) target genes. However, treatment of Lal (-/-) mice with the PPARα agonist fenofibrate further decreased plasma TG (and hepatic glucose and glycogen) concentrations in Lal (-/-) mice. Depletion of hepatic nuclear factor 4α and forkhead box protein a2 in fasted Lal (-/-) mice might be responsible for reduced expression of microsomal TG transfer protein, defective VLDL synthesis and drastically reduced plasma TG levels. CONCLUSIONS/INTERPRETATION: Our findings indicate that neither activation nor inactivation of PPARα per se but rather the availability of hepatic acyl-CoA concentrations regulates VLDL synthesis and subsequent metabolic adaptations in Lal (-/-) mice. We conclude that decreased plasma VLDL production enhances glucose uptake into skeletal muscle to compensate for the lack of energy supply.


Subject(s)
Cholesterol, VLDL/metabolism , Insulin Resistance/physiology , Sterol Esterase/metabolism , Animals , Cholesterol, VLDL/genetics , Female , Glucose/metabolism , Insulin Resistance/genetics , Lipolysis/genetics , Lipolysis/physiology , Liver/metabolism , Lysosomes/metabolism , Male , Mice , Sterol Esterase/deficiency , Sterol Esterase/genetics , Triglycerides/metabolism
11.
Anal Biochem ; 509: 130-134, 2016 09 15.
Article in English | MEDLINE | ID: mdl-27402176

ABSTRACT

(2)H2O as nonradioactive, stable marker substance is commonly used in preclinical and clinical studies and the precise determination of (2)H2O concentration in biological samples is crucial. However, aside from isotope ratio mass spectrometry (IRMS), only a very limited number of methods to accurately measure the (2)H2O concentration in biological samples are routinely established until now. In this study, we present a straightforward method to accurately measure (2)H-enrichment of rat brain interstitial fluid (ISF) and rat plasma to determine the relative recovery of a cerebral open flow microperfusion (cOFM) probe, using headspace-gas-chromatography - quadrupole-mass-spectrometry. This method is based on basic-catalyzed hydrogen/deuterium exchange in acetone and detects the (2)H-labelled acetone directly by the headspace GC-MS. Small sample volumes and limited number of preparation steps make this method highly competitive. It has been fully validated. (2)H enriched to 8800 ppm in plasma showed an accuracy of 98.9% and %Relative Standard Deviation (RSD) of 3.1 with n = 18 over three days and with two operators. Similar performance was obtained for cerebral ISF enriched to 1100 ppm (accuracy: 96.5%, %RSD: 3.1). With this highly reproducible method we demonstrated the successful employment of (2)H2O as performance marker for a cOFM probe.


Subject(s)
Brain/metabolism , Deuterium Exchange Measurement/methods , Deuterium Oxide , Extracellular Fluid , Gas Chromatography-Mass Spectrometry/methods , Animals , Deuterium Oxide/analysis , Deuterium Oxide/pharmacokinetics , Deuterium Oxide/pharmacology , Extracellular Fluid/chemistry , Extracellular Fluid/metabolism , Rats
12.
Brain Behav Immun ; 56: 140-55, 2016 Aug.
Article in English | MEDLINE | ID: mdl-26923630

ABSTRACT

Emerging evidence indicates that disruption of the gut microbial community (dysbiosis) impairs mental health. Germ-free mice and antibiotic-induced gut dysbiosis are two approaches to establish causality in gut microbiota-brain relationships. However, both models have limitations, as germ-free mice display alterations in blood-brain barrier and brain ultrastructure and antibiotics may act directly on the brain. We hypothesized that the concerns related to antibiotic-induced gut dysbiosis can only adequately be addressed if the effect of intragastric treatment of adult mice with multiple antibiotics on (i) gut microbial community, (ii) metabolite profile in the colon, (iii) circulating metabolites, (iv) expression of neuronal signaling molecules in distinct brain areas and (v) cognitive behavior is systematically investigated. Of the antibiotics used (ampicillin, bacitracin, meropenem, neomycin, vancomycin), ampicillin had some oral bioavailability but did not enter the brain. 16S rDNA sequencing confirmed antibiotic-induced microbial community disruption, and metabolomics revealed that gut dysbiosis was associated with depletion of bacteria-derived metabolites in the colon and alterations of lipid species and converted microbe-derived molecules in the plasma. Importantly, novel object recognition, but not spatial, memory was impaired in antibiotic-treated mice. This cognitive deficit was associated with brain region-specific changes in the expression of cognition-relevant signaling molecules, notably brain-derived neurotrophic factor, N-methyl-d-aspartate receptor subunit 2B, serotonin transporter and neuropeptide Y system. We conclude that circulating metabolites and the cerebral neuropeptide Y system play an important role in the cognitive impairment and dysregulation of cerebral signaling molecules due to antibiotic-induced gut dysbiosis.


Subject(s)
Anti-Bacterial Agents/adverse effects , Brain/metabolism , Cognitive Dysfunction , Colon/metabolism , Dysbiosis , Gastrointestinal Microbiome/drug effects , Recognition, Psychology , Spatial Memory , Animals , Cognitive Dysfunction/etiology , Cognitive Dysfunction/metabolism , Cognitive Dysfunction/physiopathology , Disease Models, Animal , Dysbiosis/chemically induced , Dysbiosis/complications , Dysbiosis/metabolism , Male , Metabolomics , Mice , Mice, Inbred C57BL
13.
BMC Bioinformatics ; 16: 118, 2015 Apr 16.
Article in English | MEDLINE | ID: mdl-25888443

ABSTRACT

BACKGROUND: Untargeted metabolomics generates a huge amount of data. Software packages for automated data processing are crucial to successfully process these data. A variety of such software packages exist, but the outcome of data processing strongly depends on algorithm parameter settings. If they are not carefully chosen, suboptimal parameter settings can easily lead to biased results. Therefore, parameter settings also require optimization. Several parameter optimization approaches have already been proposed, but a software package for parameter optimization which is free of intricate experimental labeling steps, fast and widely applicable is still missing. RESULTS: We implemented the software package IPO ('Isotopologue Parameter Optimization') which is fast and free of labeling steps, and applicable to data from different kinds of samples and data from different methods of liquid chromatography - high resolution mass spectrometry and data from different instruments. IPO optimizes XCMS peak picking parameters by using natural, stable (13)C isotopic peaks to calculate a peak picking score. Retention time correction is optimized by minimizing relative retention time differences within peak groups. Grouping parameters are optimized by maximizing the number of peak groups that show one peak from each injection of a pooled sample. The different parameter settings are achieved by design of experiments, and the resulting scores are evaluated using response surface models. IPO was tested on three different data sets, each consisting of a training set and test set. IPO resulted in an increase of reliable groups (146% - 361%), a decrease of non-reliable groups (3% - 8%) and a decrease of the retention time deviation to one third. CONCLUSIONS: IPO was successfully applied to data derived from liquid chromatography coupled to high resolution mass spectrometry from three studies with different sample types and different chromatographic methods and devices. We were also able to show the potential of IPO to increase the reliability of metabolomics data. The source code is implemented in R, tested on Linux and Windows and it is freely available for download at https://github.com/glibiseller/IPO . The training sets and test sets can be downloaded from https://health.joanneum.at/IPO .


Subject(s)
Algorithms , Chromatography, Liquid/methods , Electronic Data Processing/methods , Electronic Data Processing/standards , Mass Spectrometry/methods , Metabolomics/methods , Software , Animals , Carbon Radioisotopes/analysis , Heart/physiology , Humans , Lipids/analysis , Lung/metabolism , Mice , Muscles/metabolism , Programming Languages , Reproducibility of Results , Saccharomyces cerevisiae/metabolism
14.
Mol Syst Biol ; 10: 716, 2014.
Article in English | MEDLINE | ID: mdl-24489116

ABSTRACT

Lysine acetylation is a frequently occurring posttranslational modification; however, little is known about the origin and regulation of most sites. Here we used quantitative mass spectrometry to analyze acetylation dynamics and stoichiometry in Saccharomyces cerevisiae. We found that acetylation accumulated in growth-arrested cells in a manner that depended on acetyl-CoA generation in distinct subcellular compartments. Mitochondrial acetylation levels correlated with acetyl-CoA concentration in vivo and acetyl-CoA acetylated lysine residues nonenzymatically in vitro. We developed a method to estimate acetylation stoichiometry and found that the vast majority of mitochondrial and cytoplasmic acetylation had a very low stoichiometry. However, mitochondrial acetylation occurred at a significantly higher basal level than cytoplasmic acetylation, consistent with the distinct acetylation dynamics and higher acetyl-CoA concentration in mitochondria. High stoichiometry acetylation occurred mostly on histones, proteins present in histone acetyltransferase and deacetylase complexes, and on transcription factors. These data show that a majority of acetylation occurs at very low levels in exponentially growing yeast and is uniformly affected by exposure to acetyl-CoA.


Subject(s)
Acetyl Coenzyme A/genetics , Histones/genetics , Mitochondria/metabolism , Saccharomyces cerevisiae/growth & development , Acetyl Coenzyme A/metabolism , Acetylation , Histone Acetyltransferases/genetics , Histone Acetyltransferases/metabolism , Histones/chemistry , Histones/metabolism , Lysine/metabolism , Mitochondria/genetics , Protein Processing, Post-Translational , Saccharomyces cerevisiae/genetics
15.
Sports (Basel) ; 12(9)2024 Aug 28.
Article in English | MEDLINE | ID: mdl-39330709

ABSTRACT

Runner's high is a euphoric emotional state occurring during and post-physical exercise. Although previous data indicate endocannabinoids' involvement in animal runner's high, their role in human runner's high remains to be established. We investigated runner's high in healthy humans assessing mood and plasma endocannabinoid concentration changes pre- and post a 60 min outdoor run, considering sex (8 females/8 males), running frequency (4 occasional/12 regular runners) and age (median split 36 years). Mood, AEA, and 2-AG concentrations were significantly increased post-run considering all participants (p < 0.0001, p < 0.0001, p < 0.01, respectively), with 2-AG varying more than AEA concentrations. Concentrations of both endocannabinoids increased pre- to post-run in women (p < 0.01) but the AEA concentration increase was higher in females than in males (p < 0.05). Post-run concentration increase appeared to be more pronounced in occasional than in regular runners for 2-AG but not for AEA. However, regular runners experienced stronger mood increases and better post-run mood than occasional runners. Post-run endocannabinoid concentrations were increased regardless of age. AEA concentrations and their post-run changes were less affected by running frequency and age than those of 2-AG. These findings provide insights into the interplay of physical exercise, physiological/psychological factors and demographics, laying a valuable foundation for future research.

16.
Pract Lab Med ; 40: e00398, 2024 May.
Article in English | MEDLINE | ID: mdl-38745675

ABSTRACT

Aim: The metabolite α-hydroxybutyrate (α-HB) is an important marker of insulin resistance and impaired glucose tolerance allowing to identify patients at risk of developing diabetes and related metabolic disorders before any symptoms become apparent. At present, its exact quantification requires mass spectrometry (LC-MS), which is not compatible with routine laboratory use. Accordingly, a simple enzymatic-based method was assessed and its applicability and measuring accuracy compared with LC-MS was investigated. Methods: Standards, serum, and plasma samples containing α-HB were prepared with routine procedures and their α-HB contents measured with the XpressGT® enzymatic test kit photometrically or with LC-MS and multiple reaction monitoring. Results: α-HB detection with XpressGT® yielded highly linear calibration curves and 102 % recovery of stocks added to commercial samples. Stability of the analyte in serum and plasma samples prepared with various anti-coagulants was >90 % after 46 h for several widely used preparations and recovery after 3 freeze-thaw cycles was ≥95 % with these anti-coagulants. A direct comparison of 75 samples indicated very good agreement of α-HB levels determined by both methods, 86 % of XpressGT® samples being within ±20 % of LC-MS values and even 93 % within ±20 % considering only samples above 30 µM concentration. Conclusion: XpressGT®-based detection of α-HB is an easily applicable method which can be used for accurate and reliable quantification of the metabolite in clinical practice. Routine α-HB determination in patients at risk of developing diabetes would allow early establishment of preventive measures or pharmacological intervention reducing the risk for the onset of serious diabetes-related health problems.

17.
Nat Cell Biol ; 26(9): 1571-1584, 2024 Sep.
Article in English | MEDLINE | ID: mdl-39117797

ABSTRACT

Caloric restriction and intermittent fasting prolong the lifespan and healthspan of model organisms and improve human health. The natural polyamine spermidine has been similarly linked to autophagy enhancement, geroprotection and reduced incidence of cardiovascular and neurodegenerative diseases across species borders. Here, we asked whether the cellular and physiological consequences of caloric restriction and fasting depend on polyamine metabolism. We report that spermidine levels increased upon distinct regimens of fasting or caloric restriction in yeast, flies, mice and human volunteers. Genetic or pharmacological blockade of endogenous spermidine synthesis reduced fasting-induced autophagy in yeast, nematodes and human cells. Furthermore, perturbing the polyamine pathway in vivo abrogated the lifespan- and healthspan-extending effects, as well as the cardioprotective and anti-arthritic consequences of fasting. Mechanistically, spermidine mediated these effects via autophagy induction and hypusination of the translation regulator eIF5A. In summary, the polyamine-hypusination axis emerges as a phylogenetically conserved metabolic control hub for fasting-mediated autophagy enhancement and longevity.


Subject(s)
Autophagy , Caenorhabditis elegans , Caloric Restriction , Fasting , Longevity , Spermidine , Autophagy/drug effects , Longevity/drug effects , Spermidine/metabolism , Spermidine/pharmacology , Animals , Humans , Caenorhabditis elegans/metabolism , Peptide Initiation Factors/metabolism , Peptide Initiation Factors/genetics , Eukaryotic Translation Initiation Factor 5A , Drosophila melanogaster/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/genetics , Mice , Male , Mice, Inbred C57BL
18.
J Biol Chem ; 287(14): 11164-73, 2012 Mar 30.
Article in English | MEDLINE | ID: mdl-22311986

ABSTRACT

Synthesis, storage, and turnover of triacylglycerols (TAGs) in adipocytes are critical cellular processes to maintain lipid and energy homeostasis in mammals. TAGs are stored in metabolically highly dynamic lipid droplets (LDs), which are believed to undergo fragmentation and fusion under lipolytic and lipogenic conditions, respectively. Time lapse fluorescence microscopy showed that stimulation of lipolysis in 3T3-L1 adipocytes causes progressive shrinkage and almost complete degradation of all cellular LDs but without any detectable fragmentation into micro-LDs (mLDs). However, mLDs were rapidly formed after induction of lipolysis in the absence of BSA in the culture medium that acts as a fatty acid scavenger. Moreover, mLD formation was blocked by the acyl-CoA synthetase inhibitor triacsin C, implicating that mLDs are synthesized de novo in response to cellular fatty acid overload. Using label-free coherent anti-Stokes Raman scattering microscopy, we demonstrate that LDs grow by transfer of lipids from one organelle to another. Notably, this lipid transfer between closely associated LDs is not a rapid and spontaneous process but rather occurs over several h and does not appear to require physical interaction over large LD surface areas. These data indicate that LD growth is a highly regulated process leading to the heterogeneous LD size distribution within and between individual cells. Our findings suggest that lipolysis and lipogenesis occur in parallel in a cell to prevent cellular fatty acid overflow. Furthermore, we propose that formation of large LDs requires a yet uncharacterized protein machinery mediating LD interaction and lipid transfer.


Subject(s)
Adipocytes/metabolism , Lipids/chemistry , Lipolysis , 3T3-L1 Cells , Adipocytes/cytology , Animals , Cell Survival , Humans , Mice , Molecular Imaging , Stem Cells/cytology , Surface Properties , Time Factors
19.
Gastroenterology ; 142(1): 140-151.e12, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22001865

ABSTRACT

BACKGROUND & AIMS: The liver controls central processes of lipid and bile acid homeostasis. We aimed to investigate whether alterations in lipid metabolism contribute to the pathogenesis of chronic cholestatic liver disease in mice. METHODS: We used microarray and metabolic profiling analyses to identify alterations in systemic and hepatic lipid metabolism in mice with disruption of the gene ATP-binding cassette sub-family B member 4 (Abcb4(-/-) mice), a model of inflammation-induced cholestatic liver injury, fibrosis, and cancer. RESULTS: Alterations in Abcb4(-/-) mice, compared with wild-type mice, included deregulation of genes that control lipid synthesis, storage, and oxidation; decreased serum levels of cholesterol and phospholipids; and reduced hepatic long-chain fatty acyl-CoAs (LCA-CoA). Feeding Abcb4(-/-) mice the side chain-modified bile acid 24-norursodeoxycholic acid (norUDCA) reversed their liver injury and fibrosis, increased serum levels of lipids, lowered phospholipase and triglyceride hydrolase activities, and restored hepatic LCA-CoA and triglyceride levels. Additional genetic and nutritional studies indicated that lipid metabolism contributed to chronic cholestatic liver injury; crossing peroxisome proliferator-activated receptor (PPAR)-α-deficient mice with Abcb4(-/-) mice (to create double knockouts) or placing Abcb4(-/-) mice on a high-fat diet protected against liver injury, with features similar to those involved in the response to norUDCA. Placing pregnant Abcb4(-/-) mice on high-fat diets prevented liver injury in their offspring. However, fenofibrate, an activator of PPARα, aggravated liver injury in Abcb4(-/-) mice. CONCLUSIONS: Alterations in lipid metabolism contribute to the pathogenesis and progression of cholestatic liver disease in mice.


Subject(s)
Cell Proliferation , Cholestasis, Intrahepatic/metabolism , Hepatitis/metabolism , Lipid Metabolism , Liver Cirrhosis/metabolism , Liver/metabolism , ATP Binding Cassette Transporter, Subfamily B/deficiency , ATP Binding Cassette Transporter, Subfamily B/genetics , Animals , Bile Acids and Salts/metabolism , Bile Acids and Salts/pharmacology , Cholestasis, Intrahepatic/drug therapy , Cholestasis, Intrahepatic/genetics , Cholestasis, Intrahepatic/pathology , Chronic Disease , Dietary Fats/administration & dosage , Dietary Fats/metabolism , Disease Models, Animal , Disease Progression , Fatty Acids/metabolism , Female , Fenofibrate/pharmacology , Gene Expression Profiling/methods , Gene Expression Regulation , Hepatitis/drug therapy , Hepatitis/genetics , Hepatitis/pathology , Hypolipidemic Agents/pharmacology , Lipid Metabolism/genetics , Liver/drug effects , Liver/pathology , Liver Cirrhosis/drug therapy , Liver Cirrhosis/genetics , Liver Cirrhosis/pathology , Metabolomics , Mice , Mice, Knockout , Oligonucleotide Array Sequence Analysis , PPAR gamma/deficiency , PPAR gamma/genetics , Pregnancy , Prenatal Exposure Delayed Effects , Triglycerides/metabolism , Ursodeoxycholic Acid/analogs & derivatives , Ursodeoxycholic Acid/pharmacology , ATP-Binding Cassette Sub-Family B Member 4
20.
medRxiv ; 2023 Nov 29.
Article in English | MEDLINE | ID: mdl-37292870

ABSTRACT

Background: Pulmonary hypertension (PH) poses a significant health threat with high morbidity and mortality, necessitating improved diagnostic tools for enhanced management. Current biomarkers for PH lack functionality and comprehensive diagnostic and prognostic capabilities. Therefore, there is a critical need to develop biomarkers that address these gaps in PH diagnostics and prognosis. Methods: To address this need, we employed a comprehensive metabolomics analysis in 233 blood based samples coupled with machine learning analysis. For functional insights, human pulmonary arteries (PA) of idiopathic pulmonary arterial hypertension (PAH) lungs were investigated and the effect of extrinsic FFAs on human PA endothelial and smooth muscle cells was tested in vitro. Results: PA of idiopathic PAH lungs showed lipid accumulation and altered expression of lipid homeostasis-related genes. In PA smooth muscle cells, extrinsic FFAs caused excessive proliferation and endothelial barrier dysfunction in PA endothelial cells, both hallmarks of PAH.In the training cohort of 74 PH patients, 30 disease controls without PH, and 65 healthy controls, diagnostic and prognostic markers were identified and subsequently validated in an independent cohort. Exploratory analysis showed a highly impacted metabolome in PH patients and machine learning confirmed a high diagnostic potential. Fully explainable specific free fatty acid (FFA)/lipid-ratios were derived, providing exceptional diagnostic accuracy with an area under the curve (AUC) of 0.89 in the training and 0.90 in the validation cohort, outperforming machine learning results. These ratios were also prognostic and complemented established clinical prognostic PAH scores (FPHR4p and COMPERA2.0), significantly increasing their hazard ratios (HR) from 2.5 and 3.4 to 4.2 and 6.1, respectively. Conclusion: In conclusion, our research confirms the significance of lipidomic alterations in PH, introducing innovative diagnostic and prognostic biomarkers. These findings may have the potential to reshape PH management strategies.

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