GTTAtlas

metabolomický atlas myších tkání během orálního glukózového tolerančního testu (OGTT) s využitím [¹³C₆]-glukózy

• Interaktivní metabolomický, lipidomický a fluxomický atlas polárních a nepolárních metabolitů.
• Standardní orální glukózový toleranční test.
• Dvanáct metabolicky relevantních orgánů a plasma bylo analyzováno v časovém profilu OGTT.
• Šestidimenzionální LC-MS platforma byla použita na analýzu vzorků.
• Hmostnostní spektrometrie byla použita na detekci metabolitů značených ¹³C.
• Osud ¹³C-značené glokózy byl mapován na metabolické dráhy.
• Metabolické vazby uvnitř a mezi orgány byly vyhodnoceny pomocí pokročilých statistických metod.
• Software pro mapování analytů na metabolické dráhy Mapper - a pathway mapping tool.

► Magno Lopes, Kristyna Brejchova, Martin Riecan, Michaela Novakova, Martin Rossmeisl, Tomas Cajka, Ondrej Kuda^✉
Metabolomics atlas of oral 13C-glucose tolerance test in mice
Cell Reports, 2021, 27 (2), 109833 DOI https://doi.org/10.1016/j.celrep.2021.109833

Glucose tolerance represents a complex phenotype in which many tissues play important roles and interact to regulate metabolic homeostasis. Here, we perform an analysis of 13C6-glucose tissue distribution, which maps the metabolome and lipidome across 12 metabolically relevant mouse organs and plasma, with integrated 13C6-glucose-derived carbon tracing during oral glucose tolerance test (OGTT). We measure time profiles of water-soluble metabolites and lipids and integrate the global metabolite response into metabolic pathways. During the OGTT, glucose use is turned on with specific kinetics at the organ level, but fasting substrates like β-hydroxybutyrate are switched off in all organs simultaneously. Timeline profiling of 13C-labeled fatty acids and triacylglycerols across tissues suggests that brown adipose tissue may contribute to the circulating fatty acid pool at maximal plasma glucose levels. The GTTAtlas interactive web application serves as a unique resource for the exploration of whole-body glucose metabolism and time profiles of tissue and plasma metabolites during the OGTT.

• A systems-level organ-specific metabolomics atlas of mouse tissues
• ¹³C₆-glucose tracer tissue distribution during glucose tolerance test
• Interactive online application for metabolic pathway exploration
• LC-MS data resource covering polar metabolites and complex lipids

 

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MetaboAtlas21

MetaboAtlas21 je zevrubný atlas myšího metabolomu a lipidomu.

• Ve spolupráci s Laboratoří metabolomiky.
• Computational resources were supplied by the project "e-Infrastruktura CZ" (e-INFRA LM2018140) provided within the program Projects of Large Research, Development and Innovations Infrastructures.

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Mapper - a pathway mapping tool

• Online nástroj pro mapování metabolitů na metabolické mapy.
• Pomocí excelového vstupního souboru lze vizualizovat hladiny metabolitů.
• Podporuje jednoduché sloupcové grafy i složené grafy pro isotopology.

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Metabolic model viewer

• Online nástroj pro vizualizaci metabolických modelů.
• Podporuje pouze ¹³C-značené experimenty.
• Kompatibilní s MFA Suite™ (Metabolic Flux Analysis Suite) https://mfa.vueinnovations.com/.

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LORA

• Online nástroj pro Lipid Over-Representation Analysis.
• Postaven s použitím softwaru Goslin, díky čemuž podporuje různé lipidové názvosloví.
• Používá UpSet plot, circulární hierarchický strom and funkce Cytoscape network k popsání obohacení lipidomu.

► Michaela Vondrackova, Dominik Kopczynski, Nils Hoffmann, Ondrej Kuda^✉
LORA, Lipid Over-Representation Analysis Based on Structural Information
Analytical Chemistry, 2023 DOI https://doi.org/10.1021/acs.analchem.3c02039

With the increasing number of lipidomic studies, there is a need for an efficient and automated analysis of lipidomic data. One of the challenges faced by most existing approaches to lipidomic data analysis is lipid nomenclature. The systematic nomenclature of lipids contains all available information about the molecule, including its hierarchical representation, which can be used for statistical evaluation. The Lipid Over-Representation Analysis (LORA) web application (https://lora.metabolomics.fgu.cas.cz) analyzes this information using the Java-based Goslin framework, which translates lipid names into a standardized nomenclature. Goslin provides the level of lipid hierarchy, including information on headgroups, acyl chains, and their modifications, up to the "complete structure" level. LORA allows the user to upload the experimental query and reference data sets, select a grammar for lipid name normalization, and then process the data. The user can then interactively explore the results and perform lipid over-representation analysis based on selected criteria. The results are graphically visualized according to the lipidome hierarchy. The lipids present in the most over-represented terms (lipids with the highest number of enriched shared structural features) are defined as Very Important Lipids (VILs). For example, the main result of a demo data set is the information that the query is significantly enriched with "glycerophospholipids" containing "acyl 20:4" at the "sn-2 position". These terms define a set of VILs (e.g., PC 18:2/20:4;O and PE 16:0/20:4(5,8,10,14);OH). All results, graphs, and visualizations are summarized in a report. LORA is a tool focused on the smart mining of epilipidomics data sets to facilitate their interpretation at the molecular level.

• Webová aplikace LORA.metabolomics.fgu.cas.cz
• GIT https://github.com/IPHYS-Bioinformatics/LORA

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Dynamická metabolomika - stopování osudu ¹³C glukózy

5-PAHSA primes adipocytes for glucose metabolism in a different way from insulin. 5-PAHSA partially re-wires glucose metabolism pathways in favor of Krebs cycle, NADPH synthesis and de novo lipogenesis. Simplified metabolic situation is animated through the pathway within 15 minutes of glucose uptake into adipocytes. Details and conditions in Paluchova et al. DOI and FAHFA page

 

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Použití [4-²H]-glukozy k metabolickému značení NADPH.

[4-²H]-glucose can be used as a malic enzyme tracer, which specifically labels NADPH’s redox active hydrogen. The deuteron from [4-²H]-glucose is passed to NAD²H by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) during glycolysis. This cytosolic NAD²H can be used by cytosolic malate dehydrogenase 1 (MDH) to convert cytosolic oxaloacetate (coming from citrate cleavage by ATP citrate lyase) to cytosolic [2-²H]-malate. This deuterated malate can be either transported to mitochondria or converted by malic enzyme 1 (ME1) to cytosolic pyruvate and cytosolic NADP²H. This NADP²H represents the energy for DNL. During the synthesis of a fatty acid from acetyl-CoA units (via malonyl-CoA), two NADPH molecules are used per one C₂ unit. The fatty acid labeling is the result of two stochastic hydrogen selection processes, with 1/3 of hydrogens coming from water and 2/3 coming from NADPH. If cytosolic NADP²H is present, contribution of malic enzyme 1 NADPH to fatty acid synthesis can be measured.

3T3-L1 differentiated adipocytes were incubated with or without 40 µM 5-PAHSA in medium with [4-²H]-glucose for 24 hours without insulin, metabolism quenched on liquid nitrogen, cells extracted to preserve NADPH and NADP+ ratio, and raw extracts immediately measured by LC-MS/MS according to a published protocol.
Malate labeling M+1 documents that the deuteron was introduced by labeled NADH. For further details regarding malate labeling and its alternative fate see ref. Lewis et al. NADPH M+1 over NAPD+ M+1 labeling represent the redox-active deuteron on NADP²H. 5-PAHSA treatment resulted in significantly higher labeling of cytosolic NADPH. For more details see ref. Lie et al. Scheme adapted from Lewis et al.. Data from this labeling experiments were adjusted for C, H, O, N, P natural abundance and tracer purity using IsoCor-2.0.5. Details and conditions in Paluchova et al. DOI and FAHFA page

 

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Značení lipidů pomocí ²H₂O - syntéza neutrálních lipidů

For the simple synthesis of TAGs, glycerol phosphate and acyl-CoAs are needed. WAT is virtually lacking glycerol kinase, thus all glycerol phosphate has to be synthesized either via glycolysis (green path) or glyceroneogenesis (pink path). During glycolysis, deuteria (up to 3, M+3) could be incorporated from water within the equilibrium between Glyceraldehyde-Ⓟ, Dihydroxyacetone-Ⓟ, and Glycerol-3-Ⓟ. The alternative glyceroneogenic pathway precursors are deuterium-labeled within the Krebs cycle and further during conversion of Phosphoenolpyruvate to Glyceraldehyde-Ⓟ. This path may incorporate up to 5 deuteria per glycerol after several turns of the Krebs cycle. The intermediates labeled with deuteria within the Krebs cycle may also enter the de novo lipogenic pathway (blue path) as labeled Acetyl-CoA. The first (and future terminal methyl group) Acetyl-CoA keeps the deuteria, while the later Acetyl-CoAs are converted to Malonyl-CoAs and lose one deuteria from the Krebs cycle. During the elongation process of the acyl-intermediate, NADPH+H+ provides additional deuteria equilibrated from body water, thus potentially increasing the enrichment (red path). Finally, both glycerol phosphate and newly synthesized palmitate can form TAG. The degree of enrichment of the final TAG might be diluted by the contribution of FA re-esterification / lipolysis or import of external fatty acids. There are up to 6 metabolically different hydrogens, which could be exchanged for deuterium in a simple TAG.

Literature:

1. Liu et al. DOI
2. Lewis et al. DOI
3. Zhang et al. DOI
4. Krycer et al. DOI
5. Paluchova et al. DOI