Distinct metabolomic signature in cerebrospinal fluid in early parkinson's disease: Early Parkinson'S CSF Metabolic Signature

in Movement Disorders (2017)

Objective: The purpose of this study was to profile cerebrospinal fluid (CSF) from early-stage PD patients for disease-related metabolic changes and to determine a robust biomarker signature for early ... [more ▼]

Objective: The purpose of this study was to profile cerebrospinal fluid (CSF) from early-stage PD patients for disease-related metabolic changes and to determine a robust biomarker signature for early-stage PD diagnosis. Methods: By applying a non-targeted and mass spectrometry-driven approach, we investigated the CSF metabolome of 44 early-stage sporadic PD patients yet without treatment (DeNoPa cohort). We compared all detected metabolite levels with those measured in CSF of 43 age- and gender-matched healthy controls. After this analysis, we validated the results in an independent PD study cohort (T€ubingen cohort). Results: We identified that dehydroascorbic acid levels were significantly lower and fructose, mannose, and threonic acid levels were significantly higher (P <.05) in PD patients when compared with healthy controls. These changes reflect pathological oxidative stress responses, as well as protein glycation/glycosylation reactions in PD. Using a machine learning approach based on logistic regression, we successfully predicted the origin (PD patients vs healthy controls) in a second (n518) as well as in a third and completely independent validation set (n536). The biomarker signature is composed of the three markers—mannose, threonic acid, and fructose—and allows for sample classification with a sensitivity of 0.790 and a specificity of 0.800. Conclusion: We identified PD-specific metabolic changes in CSF that were associated with antioxidative stress response, glycation, and inflammation. Our results disentangle the complexity of the CSF metabolome to unravel metabolome changes related to earlystage PD. The detected biomarkers help understanding PD pathogenesis and can be applied as biomarkers to increase clinical diagnosis accuracy and patient care in early-stage PD. [less ▲]

Glutathione Primes T Cell Metabolism for Inflammation

in Immunity (2017), 46(4), 675-689

Activated T cells produce reactive oxygen species (ROS), which trigger the antioxidative glutathione (GSH) response necessary to buffer rising ROS and prevent cellular damage. We report that GSH is ... [more ▼]

Activated T cells produce reactive oxygen species (ROS), which trigger the antioxidative glutathione (GSH) response necessary to buffer rising ROS and prevent cellular damage. We report that GSH is essential for T cell effector functions through its regulation of metabolic activity. Conditional gene targeting of the catalytic subunit of glutamate cysteine ligase (Gclc) blocked GSH production specifically in murine T cells. Gclc-deficient T cells initially underwent normal activation but could not meet their increased energy and biosynthetic requirements. GSH deficiency compromised the activation of mammalian target of rapamycin-1 (mTOR) and expression of NFAT and Myc transcription factors, abrogating the energy utilization and Myc-dependent metabolic reprogramming that allows activated T cells to switch to glycolysis and glutaminolysis. In vivo, T-cell-specific ablation of murine Gclc prevented autoimmune disease but blocked antiviral defense. The antioxidative GSH pathway thus plays an unexpected role in metabolic integration and reprogramming during inflammatory T cell responses. [less ▲]

Stable Isotope-Assisted Evaluation of Different Extraction Solvents for Untargeted Metabolomics of Plants

in International Journal of Molecular Sciences (2016)

The evaluation of extraction protocols for untargeted metabolomics approaches is still difficult. We have applied a novel stable isotope-assisted workflow for untargeted LC-HRMS-based plant metabolomics ... [more ▼]

The evaluation of extraction protocols for untargeted metabolomics approaches is still difficult. We have applied a novel stable isotope-assisted workflow for untargeted LC-HRMS-based plant metabolomics , which allows for the first time every detected feature to be considered for method evaluation. The efficiency and complementarity of commonly used extraction solvents, namely 1 + 3 (v/v) mixtures of water and selected organic solvents (methanol, acetonitrile or methanol/acetonitrile 1 + 1 (v/v)), with and without the addition of 0.1% (v/v) formic acid were compared. Four different wheat organs were sampled, extracted and analysed by LC-HRMS. Data evaluation was performed with the in-house-developed MetExtract II software and R. With all tested solvents a total of 871 metabolites were extracted in ear, 785 in stem, 733 in leaf and 517 in root samples, respectively. Between 48% (stem) and 57% (ear) of the metabolites detected in a particular organ were found with all extraction mixtures, and 127 of 996 metabolites were consistently shared between all extraction agent/organ combinations. In aqueous methanol, acidification with formic acid led to pronounced pH dependency regarding the precision of metabolite abundance and the number of detectable metabolites, whereas extracts of acetonitrile-containing mixtures were less affected. Moreover, methanol and acetonitrile have been found to be complementary with respect to extraction efficiency. Interestingly, the beneficial properties of both solvents can be combined by the use of a water-methanol-acetonitrile mixture for global metabolite extraction instead of aqueous methanol or aqueous acetonitrile alone. [less ▲]

The role of HIF-1 in oncostatin M-dependent metabolic reprogramming of hepatic cells.

in Cancer and Metabolism (2016), 4

BACKGROUND: Hypoxia and inflammation have been identified as hallmarks of cancer. A majority of hepatocellular carcinomas are preceded by hepatitis B- or C-related chronic infections suggesting that liver ... [more ▼]

BACKGROUND: Hypoxia and inflammation have been identified as hallmarks of cancer. A majority of hepatocellular carcinomas are preceded by hepatitis B- or C-related chronic infections suggesting that liver cancer development is promoted by an inflammatory microenvironment. The inflammatory cytokine oncostatin M (OSM) was shown to induce the expression of hypoxia-inducible factor-1 alpha (HIF-1 alpha) under normoxic conditions in hepatocytes and hepatoma cells. HIF-1 alpha is known to orchestrate the expression of numerous genes, many of which code for metabolic enzymes that play key roles in the adaptation of cellular metabolism to low oxygen tension. RESULTS: Here, we show that OSM-induced upregulation of HIF-1 alpha reprograms cellular metabolism in three clones of the human hepatocyte cell line PH5CH (PH5CH1, PH5CH7, and PH5CH8) towards a hypoxia-like metabolic phenotype but has no significant effect on cellular metabolism of HepG2 and JHH-4 hepatoma cells. Although we observed only minor changes in glucose uptake and lactate secretion in PH5CH8 upon OSM treatment, we identified more pronounced changes in intracellular fluxes based on stable isotope labeling experiments. In particular, glucose oxidation in the tricarboxylic acid (TCA) cycle is reduced through pyruvate dehydrogenase kinase 1 (PDK1)-mediated inhibition of the pyruvate dehydrogenase complex, thereby reducing the oxidative TCA cycle flux. As a result of the impaired mitochondrial glucose and glutamine oxidation, the reductive isocitrate dehydrogenase flux was increased. CONCLUSIONS: We provide evidence that connects the inflammatory mediator OSM to a hypoxia-like metabolic phenotype. In the human hepatocyte cell line PH5CH, OSM-mediated upregulation of HIF-1 alpha and PDK1 can induce hypoxia-like metabolic changes, although to a lesser extent than hypoxia itself. Since PDK1 is overexpressed in several cancers, it might provide a causal link between chronic inflammation and malignant cellular transformation. [less ▲]

Loss of DJ-1 impairs antioxidant response by altered glutamine and serine metabolism

in Neurobiology of Disease (2016), 89

The oncogene DJ-1 has been originally identified as a suppressor of PTEN. Further on, loss-of-function mutations have been described as a causative factor in Parkinson's disease (PD). DJ-1 has an ... [more ▼]

The oncogene DJ-1 has been originally identified as a suppressor of PTEN. Further on, loss-of-function mutations have been described as a causative factor in Parkinson's disease (PD). DJ-1 has an important function in cellular antioxidant responses, but its role in central metabolism of neurons is still elusive. We applied stable isotope assisted metabolic profiling to investigate the effect of a functional loss of DJ-1 and show that DJ-1 deficient neuronal cells exhibit decreased glutamine influx and reduced serine biosynthesis. By providing precursors for GSH synthesis, these two metabolic pathways are important contributors to cellular antioxidant response. Down-regulation of these pathways, as a result of loss of DJ-1 leads to an impaired antioxidant response. Furthermore, DJ-1 deficient mouse microglia showed a weak but constitutive pro-inflammatory activation. The combined effects of altered central metabolism and constitutive activation of glia cells raise the susceptibility of dopaminergic neurons towards degeneration in patients harboring mutated DJ-1. Our work reveals metabolic alterations leading to increased cellular instability and identifies potential new intervention points that can further be studied in the light of novel translational medicine approaches. [less ▲]

Distinct Metabolic States Can Support Self-Renewal and Lipogenesis in Human Pluripotent Stem Cells under Different Culture Conditions

in Cell Reports (2016), 16(6), 1536--1547

Protein Kinase A Activation Promotes Cancer Cell Resistance to Glucose Starvation and Anoikis.

in PLoS genetics (2016), 12(3), 1005931

Cancer cells often rely on glycolysis to obtain energy and support anabolic growth. Several studies showed that glycolytic cells are susceptible to cell death when subjected to low glucose availability or ... [more ▼]

Cancer cells often rely on glycolysis to obtain energy and support anabolic growth. Several studies showed that glycolytic cells are susceptible to cell death when subjected to low glucose availability or to lack of glucose. However, some cancer cells, including glycolytic ones, can efficiently acquire higher tolerance to glucose depletion, leading to their survival and aggressiveness. Although increased resistance to glucose starvation has been shown to be a consequence of signaling pathways and compensatory metabolic routes activation, the full repertoire of the underlying molecular alterations remain elusive. Using omics and computational analyses, we found that cyclic adenosine monophosphate-Protein Kinase A (cAMP-PKA) axis activation is fundamental for cancer cell resistance to glucose starvation and anoikis. Notably, here we show that such a PKA-dependent survival is mediated by parallel activation of autophagy and glutamine utilization that in concert concur to attenuate the endoplasmic reticulum (ER) stress and to sustain cell anabolism. Indeed, the inhibition of PKA-mediated autophagy or glutamine metabolism increased the level of cell death, suggesting that the induction of autophagy and metabolic rewiring by PKA is important for cancer cellular survival under glucose starvation. Importantly, both processes actively participate to cancer cell survival mediated by suspension-activated PKA as well. In addition we identify also a PKA/Src mechanism capable to protect cancer cells from anoikis. Our results reveal for the first time the role of the versatile PKA in cancer cells survival under chronic glucose starvation and anoikis and may be a novel potential target for cancer treatment. [less ▲]

Isotopologue ratio normalization for non-targeted metabolomics

in Journal of Chromatography. A (2015), 1389

Robust quantification of analytes is a prerequisite for meaningful metabolomics experiments. In non-targeted metabolomics it is still hard to compare measurements across multiple batches or instruments ... [more ▼]

Robust quantification of analytes is a prerequisite for meaningful metabolomics experiments. In non-targeted metabolomics it is still hard to compare measurements across multiple batches or instruments. For targeted analyses isotope dilution mass spectrometry is used to provide a robust normalization reference. Here, we present an approach that allows for the automated semi-quantification of metabolites relative to a fully stable isotope-labeled metabolite extract. Unlike many previous approaches, we include both identified and unidentified compounds in the data analysis. The internal standards are detected in an automated manner using the non-targeted tracer fate detection algorithm. The ratios of the light and heavy form of these compounds serve as a robust measure to compare metabolite levels across different mass spectrometric platforms. As opposed to other methods which require high resolution mass spectrometers, our methodology works with low resolution mass spectrometers as commonly used in gas chromatography electron impact mass spectrometry (GC–EI-MS)-based metabolomics. We demonstrate the validity of our method by analyzing compound levels in different samples and show that it outperforms conventional normalization approaches in terms of intra- and inter-instrument reproducibility. We show that a labeled yeast metabolite extract can also serve as a reference for mammalian metabolite extracts where complete stable isotope labeling is hard to achieve. [less ▲]

How metabolites modulate metabolic flux

in Current Opinion in Biotechnology (2015), 34

Adaptation to metabolic needs and changing environments is a basic requirement of every living system. These adaptations can be very quick and mild or slower but more drastic. In any case, cells have to ... [more ▼]

Adaptation to metabolic needs and changing environments is a basic requirement of every living system. These adaptations can be very quick and mild or slower but more drastic. In any case, cells have to constantly monitor their metabolic state and requirements.In this article we review general concepts as well as recent advances on how metabolites can regulate metabolic fluxes. We discuss how cells sense metabolite levels and how changing metabolite levels regulate metabolic enzymes on different levels, from specific allosteric regulation to global transcriptional regulation. We thereby focus on local metabolite sensing in mammalian cells and show that several major discoveries have only very recently been made. [less ▲]

A roadmap for interpreting (13)C metabolite labeling patterns from cells.

in Current opinion in biotechnology (2015), 34

Measuring intracellular metabolism has increasingly led to important insights in biomedical research. (13)C tracer analysis, although less information-rich than quantitative (13)C flux analysis that ... [more ▼]

Measuring intracellular metabolism has increasingly led to important insights in biomedical research. (13)C tracer analysis, although less information-rich than quantitative (13)C flux analysis that requires computational data integration, has been established as a time-efficient method to unravel relative pathway activities, qualitative changes in pathway contributions, and nutrient contributions. Here, we review selected key issues in interpreting (13)C metabolite labeling patterns, with the goal of drawing accurate conclusions from steady state and dynamic stable isotopic tracer experiments. [less ▲]