Global 13C tracing and metabolic flux analysis of intact human liver tissue ex vivo
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PROVIDER: PRJNA1129474 | ENA |
REPOSITORIES: ENA
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Project description:Liver metabolism is central to human physiology and influences the pathogenesis of common metabolic diseases. Yet, our understanding of human liver metabolism remains incomplete, with much of current knowledge based on animal or cell culture models that do not fully recapitulate human physiology. Here, we performed in-depth measurement of metabolism in intact human liver tissue ex vivo using global 13C tracing, non-targeted mass spectrometry and model-based metabolic flux analysis. Cultured liver tissue exhibited normal anatomical structure and retained canonical liver functions such as glucose production, albumin and VLDL synthesis at near-physiological rates. Isotope tracing with a highly 13C-labeled medium generated 13C enrichment in hundreds of compounds, allowing qualitative assessment of a wide range of metabolic pathways within a single experiment. This confirmed well-known features of liver metabolism, but also revealed unexpected metabolic activities such as de novo creatine synthesis and branched-chain amino acid transamination, where human liver appears to differ from rodent models. Metabolic flux analysis identified glycogenolysis as the main source of glucose production, which could be suppressed by pharmacological inhibition of glycogen phosphorylase. Glucose production ex vivo also correlated with donor plasma glucose, suggesting that cultured liver tissue retains individual metabolic phenotypes. Moreover, liver tissue responded to postprandial levels of nutrients and insulin by suppressing glucose production and increasing nutrient uptake. Isotope tracing ex vivo allows measuring human liver metabolism with great depth and resolution in an experimentally tractable system.
2024-07-08 | GSE271041 | GEO
Project description:Liver metabolism is central to human physiology and influences the pathogenesis of common metabolic diseases. Yet, our understanding of human liver metabolism remains incomplete, with much of current knowledge based on animal or cell culture models that do not fully recapitulate human physiology. Here, we performed in-depth measurement of metabolism in intact human liver tissue ex vivo using global 13C tracing, non-targeted mass spectrometry and model-based metabolic flux analysis. Cultured liver tissue exhibited normal anatomical structure and retained canonical liver functions such as glucose production, albumin and VLDL synthesis at near-physiological rates. Isotope tracing with a highly 13C-labeled medium generated 13C enrichment in hundreds of compounds, allowing qualitative assessment of a wide range of metabolic pathways within a single experiment. This confirmed well-known features of liver metabolism, but also revealed unexpected metabolic activities such as de novo creatine synthesis and branched-chain amino acid transamination, where human liver appears to differ from rodent models. Metabolic flux analysis identified glycogenolysis as the main source of glucose production, which could be suppressed by pharmacological inhibition of glycogen phosphorylase. Glucose production ex vivo also correlated with donor plasma glucose, suggesting that cultured liver tissue retains individual metabolic phenotypes. Moreover, liver tissue responded to postprandial levels of nutrients and insulin by suppressing glucose production and increasing nutrient uptake. Isotope tracing ex vivo allows measuring human liver metabolism with great depth and resolution in an experimentally tractable system.
2024-07-23 | MTBLS10481 | MetaboLights
Project description:Cross-feeding is fundamental to the diversity and function of microbial communities. However, identification of cross-fed metabolites is often challenging due to the universality of metabolic and biosynthetic intermediates. Here, we use 13C isotope tracing in peptides to elucidate cross-fed metabolites in cocultures of Saccharomyces cerevisiae and Lactococcus lactis. The community was grown on lactose as the main carbon source with either glucose or galactose fraction of the molecule labelled with 13C. Data analysis allowing for the possible mass-shifts yielded hundreds of peptides for which we could assign both species identity and labelling degree. The labelling pattern showed that the yeast utilized galactose and, to a lesser extent, lactic acid shared by L. lactis as carbon sources. While the yeast provided essential amino acids to the bacterium as expected, the data also uncovered a complex pattern of amino acid exchange. The identity of the cross-fed metabolites was further supported by metabolite labelling in the co-culture supernatant, and by diminished fitness of a galactose-negative yeast mutant in the community. Together, our results demonstrate the utility of 13C-based proteomics for uncovering microbial interactions.
2023-02-07 | PXD019952 | Pride
Project description:By fermenting dietary fiber, the gut microbiota supplies carbon to host epithelial cells in the form of short-chain fatty acids and other metabolic byproducts. To track the transfer of carbon from fiber to host tissues via the microbiota and more clearly define the molecules mediating this transfer, we conducted stable isotope tracing in mice with U-13C-labeled inulin followed by untargeted metabolomics by LC-MS. Additionally, we applied this labeling approach to mice with chemically induced colitis to examine how inflammation impacts carbon transfer from the microbiota to host tissues, which may aid in understanding the development of inflammatory bowel diseases.
2021-07-04 | MSV000087752 | MassIVE
Project description:Primary Mouse hepatocytes were introduced to U-13C glucose or U-13C glutamine for 30 min and then analyzed by GC-MS and LC-MS.
2019-06-03 | MSV000083885 | MassIVE
Project description:Metagenomics of 13C-enriched estrogens-contaminated soil
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Project description:Deciphering the mechanisms of regulation of metabolic networks subjected to perturbations, including disease states and drug-induced stress, relies on tracing metabolic fluxes. One of the most informative data to predict metabolic fluxes are 13C based metabolomics, which provide information about how carbons are redistributed along central carbon metabolism. Such data can be integrated using 13C Metabolic Flux Analysis (13C MFA) to provide quantitative metabolic maps of flux distributions. However, 13C MFA might be unable to reduce the solution space towards a unique solution either in large metabolic networks or when small sets of measurements are integrated. Here we present parsimonious 13C MFA (p13CMFA), an approach that runs a secondary optimization in the 13C MFA solution space to identify the solution that minimizes the total reaction flux. Furthermore, flux minimization can be weighted by gene expression measurements allowing seamless integration of gene expression data with 13C data. As proof of concept, we demonstrate how p13CMFA can be used to estimate intracellular flux distributions from 13C measurements and transcriptomics data. We have implemented p13CMFA in Iso2Flux, our in-house developed isotopic steady-state 13C MFA software. The source code is freely available on GitHub (https://github.com/cfoguet/iso2flux/releases/tag/0.7.2).
| S-EPMC6750616 | biostudies-literature
Project description:By integration of transcriptome, CHIP-seq, ATAC-seq, proteomic and metabolite screening followed by carbon tracing (U-13C-Glucose, U-13C-Glutamine and U-13C-Palmitic acid) and extracellular flux analysis we provided evidence that genetic (shRNA and CRISPR/Cas9) and pharmacological (Alisertib) AURKA inhibition elicited substantial metabolic reprogramming supported in part by inhibition of MYC targets and concomitant activation of PPARA signaling. While glycolysis was suppressed by AURKA inhibition, we noted a compensatory increase in oxygen consumption rate fueled by enhanced fatty acid oxidation (FAO). Whereas interference with AURKA elicited a suppression of c-Myc, we detected an upregulation of PGC1α, a master regulator of oxidative metabolism, upon AURKA inhibition. Chromatin immunoprecipitation experiments confirmed binding of c-Myc to the promoter region of PGC1α, which is abrogated by AURKA inhibition and in turn unleashed PGC1α expression. To interfere with this oxidative metabolic reprogramming, we combined AURKA inhibitors with inhibitors of FAO (etomoxir) and electron transport chain (gamitrinib) and found substantial synergistic growth inhibition in patient derived xenograft in vitro and extension of overall survival without induction of toxicity in normal tissue.
2021-06-01 | GSE161573 | GEO
Project description:By integration of transcriptome, CHIP-seq, ATAC-seq, proteomic and metabolite screening followed by carbon tracing (U-13C-Glucose, U-13C-Glutamine and U-13C-Palmitic acid) and extracellular flux analysis we provided evidence that genetic (shRNA and CRISPR/Cas9) and pharmacological (Alisertib) AURKA inhibition elicited substantial metabolic reprogramming supported in part by inhibition of MYC targets and concomitant activation of PPARA signaling. While glycolysis was suppressed by AURKA inhibition, we noted a compensatory increase in oxygen consumption rate fueled by enhanced fatty acid oxidation (FAO). Whereas interference with AURKA elicited a suppression of c-Myc, we detected an upregulation of PGC1α, a master regulator of oxidative metabolism, upon AURKA inhibition. Chromatin immunoprecipitation experiments confirmed binding of c-Myc to the promoter region of PGC1α, which is abrogated by AURKA inhibition and in turn unleashed PGC1α expression. To interfere with this oxidative metabolic reprogramming, we combined AURKA inhibitors with inhibitors of FAO (etomoxir) and electron transport chain (gamitrinib) and found substantial synergistic growth inhibition in patient derived xenograft in vitro and extension of overall survival without induction of toxicity in normal tissue.
2021-06-01 | GSE161572 | GEO
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| 2410037 | ecrin-mdr-crc