Software:

GTTAtlas

MetaboAtlas21

Mapper

Reaction network viewer

LORA

Methodological highlighs from publications using small molecule omics techniques.

GTTAtlas

Metabolomics atlas of mouse tissue during oral glucose tolerance test using [¹³C₆]-glucose as a tracer.

• Interactive metabolomics and lipidomics atlas of polar and non-polar metabolites.
• Standard oral glucose tolerance test (OGTT) with [¹³C₆]-glucose was performed.
• Twelve metabolically-relevant mouse organs and plasma were collected at specific time points during the OGTT.
• Six-dimensional metabolipidomic LC-MS platform was used to analyze samples.
• High resolution accurate mass spectrometry was used to detect metabolite isotopologues labeled with ¹³C.
• Metabolic pathways were used to explore the fate of ¹³C-labeled glucose.
• Inter-organ metabolism of glucose and its metabolites was evaluated.
• Software for metabolic pathway mapping: 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 …

• 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

 MetaboAtlas21

MetaboAtlas21 is a comprehensive atlas of the mouse metabolome and lipidome.

• In collaboration with Laboratory of Metabolomics.
• Interactive metabolomics and lipidomics atlas of polar and non-polar metabolites.
• Twenty one mouse organs and biofluids & two diets & three reference serum/plasma samples were analyzed.
• Seven/eight-dimensional metabolipidomic LC-MS platform was used to analyze samples.
• 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.

 Mapper – a pathway mapping tool

• Online metabolite mapping tool.
• Use an excel file to map metabolite concentration on a metabolic pathway map.
• Supports simple bar graphs and stacked bar graphs for isotopologues.

 Metabolic model viewer

• Online metabolic network (biochemical reaction network) visualization tool.
• Supports ¹³C-based models and elementary metabolite unit (EMU) notation ‘A (abc) -> B (ab) + C (c)’.
• Compatible with models for the MFA Suite™ (Metabolic Flux Analysis Suite) https://mfa.vueinnovations.com/.

 LORA

• • Online Lipid Over-Representation Analysis tool.
  • Built on Goslin grammars, supports multiple lipid annotation dialects.
  • Uses UpSet plot, circular hierarchical tree and Cytoscape network to describe lipidome data enrichment.

  ► 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……

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

Dynamic metabolomics – tracing ¹³C glucose

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

Use of [4-²H]-glucose for metabolic labeling of 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

Lipid labeling using ²H₂O – labeling of neutral lipids

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