scholarly journals Metabolomics and lipidomics in C. elegans using a single sample preparation

2021 ◽  
pp. dmm.047746
Author(s):  
Marte Molenaars ◽  
Bauke V. Schomakers ◽  
Hyung L. Elfrink ◽  
Arwen W. Gao ◽  
Martin A. T. Vervaart ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Model Organism ◽  
Tca Cycle ◽  
Single Sample ◽  
Biologically Relevant ◽  
C Elegans ◽  
The Tca Cycle ◽  
Powerful Analytical Tool ◽  
Increasing Demand ◽  
Data Yield

Comprehensive metabolomic and lipidomic mass spectrometry methods are in increasing demand, for instance in research related to nutrition and aging. The nematode C. elegans is a key model organism in these fields, due to the large repository of available C. elegans mutants and their convenient natural lifespan. Here, we describe a robust and sensitive analytical method for the semi-quantitative analysis of >100 polar (metabolomics) and >1000 apolar (lipidomics) metabolites in C. elegans, using a single sample preparation. Our method is capable of reliably detecting a wide variety of biologically relevant metabolic aberrations in, for instance, glycolysis and the TCA cycle, pyrimidine metabolism and complex lipid biosynthesis. In conclusion, we provide a powerful analytical tool that maximizes metabolic data yield from a single sample.

scholarly journals Metabolomics and lipidomics in C. elegans using a single sample preparation

2020 ◽  
Author(s):  
Marte Molenaars ◽  
Bauke V. Schomakers ◽  
Hyung L. Elfrink ◽  
Arwen W. Gao ◽  
Martin A.T. Vervaart ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Model Organism ◽  
Tca Cycle ◽  
Single Sample ◽  
Biologically Relevant ◽  
C Elegans ◽  
The Tca Cycle ◽  
Powerful Analytical Tool ◽  
Increasing Demand ◽  
Data Yield

AbstractComprehensive metabolomic and lipidomic mass spectrometry methods are in increasing demand, for instance in research related to nutrition and aging. The nematode C. elegans is a key model organism in these fields, due to the large repository of available C. elegans mutants and their convenient natural lifespan. Here, we describe a robust and sensitive analytical method for the semi-quantitative analysis of >100 polar (metabolomics) and >1000 apolar (lipidomics) metabolites in C. elegans, using a single sample preparation. Our method is capable of reliably detecting a wide variety of biologically relevant metabolic aberrations in, for instance, glycolysis and the TCA cycle, pyrimidine metabolism and complex lipid biosynthesis. In conclusion, we provide a powerful analytical tool that maximizes metabolic data yield from a single sample.Graphical abstract

scholarly journals Adrenal Hormone Interactions and Metabolism: A Single Sample Multi-Omics Approach

2021 ◽  
Author(s):  
Nicole Bechmann ◽  
Deepika Watts ◽  
Charlotte Steenblock ◽  
Paal William Wallace ◽  
Annette Schürmann ◽  
...  
Keyword(s):  
Chromaffin Cells ◽  
Tca Cycle ◽  
Single Sample ◽  
Adrenal Hormones ◽  
Hormone Interactions ◽  
Adrenal Hormone ◽  
Native Proteins ◽  
The Tca Cycle ◽  
Related Enzyme ◽  
Improved Methods

AbstractThe adrenal gland is important for many physiological and pathophysiological processes, but studies are often restricted by limited availability of sample material. Improved methods for sample preparation are needed to facilitate analyses of multiple classes of adrenal metabolites and macromolecules in a single sample. A procedure was developed for preparation of chromaffin cells, mouse adrenals, and human chromaffin tumors that allows for multi-omics analyses of different metabolites and preservation of native proteins. To evaluate the new procedure, aliquots of samples were also prepared using conventional procedures. Metabolites were analyzed by liquid-chromatography with mass spectrometry or electrochemical detection. Metabolite contents of chromaffin cells and tissues analyzed with the new procedure were similar or even higher than with conventional methods. Catecholamine contents were comparable between both procedures. The TCA cycle metabolites, cis-aconitate, isocitate, and α-ketoglutarate were detected at higher concentrations in cells, while in tumor tissue only isocitrate and potentially fumarate were measured at higher contents. In contrast, in a broad untargeted metabolomics approach, a methanol-based preparation procedure of adrenals led to a 1.3-fold higher number of detected metabolites. The established procedure also allows for simultaneous investigation of adrenal hormones and related enzyme activities as well as proteins within a single sample. This novel multi-omics approach not only minimizes the amount of sample required and overcomes problems associated with tissue heterogeneity, but also provides a more complete picture of adrenal function and intra-adrenal interactions than previously possible.

scholarly journals Arsenate induced changes in bacterial metabolite and lipid pools during phosphate stress

2020 ◽  
Author(s):  
Weiping Zhuang ◽  
Narayanaganesh Balasubramanian ◽  
Lu Wang ◽  
Qian Wang ◽  
Timothy R. McDermott ◽  
...  
Keyword(s):  
Environmental Fate ◽  
Environmental Pollutants ◽  
Model Organism ◽  
Tca Cycle ◽  
Resistance Mechanisms ◽  
Intracellular Metabolite ◽  
The Tca Cycle ◽  
Arsenic Detoxification ◽  
Induced Changes ◽  
The Impact

Agrobacterium tumefaciens GW4 is a heterotrophic arsenite oxidizing bacterium with a high resistance to arsenic toxicity. It is now a model organism for studying the processes of arsenic detoxification and utilization. Previously, we demonstrated that under low phosphate conditions, arsenate (As(V)) could enhance bacterial growth and be incorporated into biomolecules including lipids. While the basic microbial As(V) resistance mechanisms have been characterized, global metabolic responses under low phosphate remain largely unknown. In the present work, the impact of As(V) and low phosphate on intracellular metabolite and lipid profiles of GW4 were quantified using liquid chromatography-mass spectroscopy (LC-MS) in combination with transcriptional assays and the analysis of intracellular ATP and NADH levels. Metabolite profiling revealed that oxidative stress response pathways were altered and suggested an increase in DNA repair. Changes in metabolite levels in the TCA cycle along with increased ATP are consistent with As(V)-enhanced growth of A. tumefaciens GW4. Lipidomics analysis revealed that most glycerophospholipids decreased in abundance when As(V) was available. However, several glycerolipid classes increased, an outcome that is consistent with maximizing growth via a phosphate sparing phenotype. Differentially regulated lipids included phosphotidylcholine and lysophospholipids, which have not been previously reported in A. tumefaciens. The metabolites and lipids identified in this study deepen our understanding of the interplay between phosphate and arsenate on chemical and metabolic levels. IMPORTANCE Arsenic is widespread in the environment and is one of the most ubiquitous environmental pollutants. Parodoxically, the growth of certain bacteria is enhanced by arsenic when phosphate is limited. Arsenate and phosphate are chemically similar and this behavior is believed to represent a phosphate sparing phenotype in which arsenate is used in place of phospohate in certain biomolecules. The research presented here uses a global approach to track metabolic changes in an environmentally relevant bacteria during exposure to arsenate when phosphate is low. Our findings are relevant for understanding the environmental fate of arsenic as well as how human associated microbiomes respond to this common toxin.

scholarly journals An insulin, AMPK, and steroid hormone-mediated metabolic switch regulates the transition between growth and diapause in C. elegans

2018 ◽  
Author(s):  
Sider Penkov ◽  
Bharath Kumar Raghuraman ◽  
Cihan Erkut ◽  
Jana Oertel ◽  
Roberta Galli ◽  
...  
Keyword(s):  
Steroid Hormone ◽  
Tca Cycle ◽  
Branch Points ◽  
Metabolic Switch ◽  
Regulatory Pathways ◽  
C Elegans ◽  
Molar Ratios ◽  
Dauer Larva ◽  
The Tca Cycle ◽  
Metabolic Transition

AbstractThe balance between growth and quiescence depends on the global metabolic state. The dauer larva of C. elegans, a developmentally arrested stage for survival under adverse environment, undergoes a major metabolic transition. Here, we show that this switch involves the concerted activity of several regulatory pathways. Whereas the steroid hormone receptor DAF-12 controls dauer morphogenesis, the insulin pathway maintains low energy expenditure through DAF-16/FoxO, which also requires AAK-2/AMPKα. DAF-12 and AAK-2 separately promote a shift in the molar ratios between competing enzymes at two key branch points within the central carbon metabolic pathway. This way, carbon atoms are diverted from the TCA cycle and directed to gluconeogenesis. When both AAK-2 and DAF-12 are suppressed, the TCA cycle is active and the developmental arrest is bypassed. Hence, the metabolic status of each developmental stage is defined by stoichiometric ratios within the constellation of metabolic enzymes and controls the transition between growth and quiescence.

Liquid-liquid extraction of succinate using a dicopper cryptate

2020 ◽  
Author(s):  
Riccardo Mobili ◽  
Sonia La Cognata ◽  
Francesca Merlo ◽  
Andrea Speltini ◽  
Massimo Boiocchi ◽  
...  
Keyword(s):  
Aqueous Phase ◽  
Visible Spectrum ◽  
Tca Cycle ◽  
Residual Concentration ◽  
Waste Products ◽  
Liquid Liquid Extraction ◽  
The Tca Cycle ◽  
Quantitative Extraction ◽  
Uv Visible

<div> <p>The extraction of the succinate dianion from a neutral aqueous solution into dichloromethane is obtained using a lipophilic cage-like dicopper(II) complex as the extractant. The quantitative extraction exploits the high affinity of the succinate anion for the cavity of the azacryptate. The anion is effectively transferred from the aqueous phase, buffered at pH 7 with HEPES, into dichloromethane. A 1:1 extractant:anion adduct is obtained. Extraction can be easily monitored by following changes in the UV-visible spectrum of the dicopper complex in dichloromethane, and by measuring the residual concentration of succinate in the aqueous phase by HPLC−UV. Considering i) the relevance of polycarboxylates in biochemistry, as e.g. normal intermediates of the TCA cycle, ii) the relevance of dicarboxylates in the environmental field, as e.g. waste products of industrial processes, and iii) the recently discovered role of succinate and other dicarboxylates in pathophysiological processes including cancer, our results open new perspectives for research in all contexts where selective recognition, trapping and extraction of polycarboxylates is required. </p> </div>

scholarly journals Mitochondrial Mistranslation in Brain Provokes a Metabolic Response Which Mitigates the Age-Associated Decline in Mitochondrial Gene Expression

2021 ◽  
Vol 22 (5) ◽  
pp. 2746
Author(s):  
Dimitri Shcherbakov ◽  
Reda Juskeviciene ◽  
Adrián Cortés Sanchón ◽  
Margarita Brilkova ◽  
Hubert Rehrauer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Metabolic Response ◽  
Mitochondrial Gene ◽  
Tca Cycle ◽  
Brain Mitochondria ◽  
Mitochondrial Gene Expression ◽  
Rna Seq ◽  
The Tca Cycle ◽  
Neurological Phenotype

Mitochondrial misreading, conferred by mutation V338Y in mitoribosomal protein Mrps5, in-vivo is associated with a subtle neurological phenotype. Brain mitochondria of homozygous knock-in mutant Mrps5V338Y/V338Y mice show decreased oxygen consumption and reduced ATP levels. Using a combination of unbiased RNA-Seq with untargeted metabolomics, we here demonstrate a concerted response, which alleviates the impaired functionality of OXPHOS complexes in Mrps5 mutant mice. This concerted response mitigates the age-associated decline in mitochondrial gene expression and compensates for impaired respiration by transcriptional upregulation of OXPHOS components together with anaplerotic replenishment of the TCA cycle (pyruvate, 2-ketoglutarate).

scholarly journals Metabolomics of aging in primary fibroblasts from small and large breed dogs

GeroScience ◽  
2021 ◽  
Author(s):  
Paul S. Brookes ◽  
Ana Gabriela Jimenez
Keyword(s):  
Aging Process ◽  
Tca Cycle ◽  
Therapeutic Strategies ◽  
Targeted Metabolomics ◽  
The Tca Cycle ◽  
Age Dependent ◽  
Primary Fibroblasts ◽  
Aging Rates ◽  
Underlying Mechanisms ◽  
Coenzyme A Biosynthesis

AbstractAmong several animal groups (eutherian mammals, birds, reptiles), lifespan positively correlates with body mass over several orders of magnitude. Contradicting this pattern are domesticated dogs, with small dog breeds exhibiting significantly longer lifespans than large dog breeds. The underlying mechanisms of differing aging rates across body masses are unclear, but it is generally agreed that metabolism is a significant regulator of the aging process. Herein, we performed a targeted metabolomics analysis on primary fibroblasts isolated from small and large breed young and old dogs. Regardless of size, older dogs exhibited lower glutathione and ATP, consistent with a role for oxidative stress and bioenergetic decline in aging. Furthermore, several size-specific metabolic patterns were observed with aging, including the following: (i) An apparent defect in the lower half of glycolysis in large old dogs at the level of pyruvate kinase. (ii) Increased glutamine anaplerosis into the TCA cycle in large old dogs. (iii) A potential defect in coenzyme A biosynthesis in large old dogs. (iv) Low nucleotide levels in small young dogs that corrected with age. (v) An age-dependent increase in carnitine in small dogs that was absent in large dogs. Overall, these data support the hypothesis that alterations in metabolism may underlie the different lifespans of small vs. large breed dogs, and further work in this area may afford potential therapeutic strategies to improve the lifespan of large dogs.

scholarly journals The Metabolic Fates of Pyruvate in Normal and Neoplastic Cells

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 762
Author(s):  
Edward V. Prochownik ◽  
Huabo Wang
Keyword(s):  
Metabolic Pathways ◽  
Redox State ◽  
Pyruvate Dehydrogenase Complex ◽  
Tca Cycle ◽  
Vascular Supply ◽  
Extrinsic Factors ◽  
The Tca Cycle ◽  
Initial Substrate ◽  
Numerous Cell ◽  
Bio Synthesis

Pyruvate occupies a central metabolic node by virtue of its position at the crossroads of glycolysis and the tricarboxylic acid (TCA) cycle and its production and fate being governed by numerous cell-intrinsic and extrinsic factors. The former includes the cell’s type, redox state, ATP content, metabolic requirements and the activities of other metabolic pathways. The latter include the extracellular oxygen concentration, pH and nutrient levels, which are in turn governed by the vascular supply. Within this context, we discuss the six pathways that influence pyruvate content and utilization: 1. The lactate dehydrogenase pathway that either converts excess pyruvate to lactate or that regenerates pyruvate from lactate for use as a fuel or biosynthetic substrate; 2. The alanine pathway that generates alanine and other amino acids; 3. The pyruvate dehydrogenase complex pathway that provides acetyl-CoA, the TCA cycle’s initial substrate; 4. The pyruvate carboxylase reaction that anaplerotically supplies oxaloacetate; 5. The malic enzyme pathway that also links glycolysis and the TCA cycle and generates NADPH to support lipid bio-synthesis; and 6. The acetate bio-synthetic pathway that converts pyruvate directly to acetate. The review discusses the mechanisms controlling these pathways, how they cross-talk and how they cooperate and are regulated to maximize growth and achieve metabolic and energetic harmony.

scholarly journals Health and longevity studies in C. elegans: the “healthy worm database” reveals strengths, weaknesses and gaps of test compound-based studies

2021 ◽  
Vol 22 (2) ◽  
pp. 215-236
Author(s):  
Nadine Saul ◽  
Steffen Möller ◽  
Francesca Cirulli ◽  
Alessandra Berry ◽  
Walter Luyten ◽  
...  
Keyword(s):  
Healthy Aging ◽  
Model Organism ◽  
Research Field ◽  
Aging Research ◽  
Genetic Manipulations ◽  
C Elegans ◽  
Starting Point ◽  
Health Related ◽  
Phenotype Measurement ◽  
Or Gene

AbstractSeveral biogerontology databases exist that focus on genetic or gene expression data linked to health as well as survival, subsequent to compound treatments or genetic manipulations in animal models. However, none of these has yet collected experimental results of compound-related health changes. Since quality of life is often regarded as more valuable than length of life, we aim to fill this gap with the “Healthy Worm Database” (http://healthy-worm-database.eu). Literature describing health-related compound studies in the aging model Caenorhabditis elegans was screened, and data for 440 compounds collected. The database considers 189 publications describing 89 different phenotypes measured in 2995 different conditions. Besides enabling a targeted search for promising compounds for further investigations, this database also offers insights into the research field of studies on healthy aging based on a frequently used model organism. Some weaknesses of C. elegans-based aging studies, like underrepresented phenotypes, especially concerning cognitive functions, as well as the convenience-based use of young worms as the starting point for compound treatment or phenotype measurement are discussed. In conclusion, the database provides an anchor for the search for compounds affecting health, with a link to public databases, and it further highlights some potential shortcomings in current aging research.

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