Introduction: Tracer-based metabolic profiling using isotope-enriched feedstocks for NMR-focussed studies in principle allows the metabolic fate of key metabolic precursors to be tracked. The liver is involved in many metabolic processes. Non-alcoholic fatty liver disease (NAFLD) is a common health concern these days which is marked by lipid accumulation within hepatocytes (Hepatic steatosis). A metabolomics study of liver steatosis holds the potential for understanding the mechanistic drivers or diagnostic markers that could enable the longer-term development of personalized medicine strategies. Analytical methods including NMR spectroscopy, have not yet been applied to such a study, thereby creating an open field for research investigation.
Aims: To assign a product to one particular or multiple metabolic pathways that are altered in hepatic steatosis by the use of NMR with the aid of different 13C-Labeled energetic compounds. To optimize a protocol that is suitable for NMR-based metabolomic study using an in vitro model of steatosis. To evaluate some NMR pulse sequences that could provide benefit in the context of NMR-based metabolomics as well as to investigate the analytical power of the 600 MHz spectrometer.
Methodology: 3-13C-Lactate, 13C3-pyruvate, and 13C8-octanoate have been utilized to induce steatosis in HepaRGTM cells. Following a specified treatment period, an optimized protocol involving quenching, harvesting, cell lysis, and extraction was implemented to isolate the intracellular components of the HepaRGTM cells. Each step of this protocol was meticulously refined during the initial stages of the project to ensure the highest efficiency and reproducibility. This optimized protocol was subsequently applied to generate metabolomics data.
The exploratory NMR metabolomics analysis enabled the identification of both exo- and endo-metabolites, including lactate, alanine, branched-chain amino acids, TCA cycle intermediates (such as succinate and citrate), triglycerides, and linoleic acids. These metabolites provided preliminary insights into the biological behavior of potential therapeutic agents for steatosis.
The analytical performance of the 600 MHz spectrometer, which served as the primary site of work, was evaluated through a comprehensive limit of detection (LOD) and limit of quantification (LOQ) procedure using a standard sample 13C18-oleic acid. Additionally, the benefits of employing a higher magnetic field with a cooled probe (800 MHz) were also assessed to determine its usefulness. Moreover, various NMR pulse sequences were tested and evaluated throughout the project to determine their effectiveness in tracing the fate of carbon-13 enrichment. Numerous samples were prepared and analyzed using NMR spectroscopy. Subsequent data processing and statistical analyses were conducted to identify key metabolites and metabolic pathways that underwent significant changes. To monitor fat deposits within HepaRGTM cells following incubation with lipogenic compounds, Oil Red O staining was employed. This staining technique helped to visualize and quantify lipid accumulation, thereby supporting the assessment of steatosis and the effectiveness of the lipogenic treatments.
Results: By utilizing different NMR pulse sequences, it was found that key lipid classes such as triglycerides (which contain unsaturated fatty acids), lactate, alanine, and intermediates within the tricarboxylic acid (TCA) cycle (such as succinate) are associated with the progression of steatosis. This discovery aids in identifying new therapeutic targets for treating steatosis. Metabolic pathways related to lipolysis, gluconeogenesis, branched-chain amino acid synthesis, and triglyceride biosynthesis were found to differ significantly after incubating HepaRGTM cells with lipogenic compounds.
The optimized protocol for this study includes the following main steps: (i) growth and differentiation of the cell culture, (ii) quenching in precooled methanol: water, (iii) cell harvesting using scraping, (iv) cell lysis by freeze-thaw cycles (three cycles), and (v) a dual phase extraction procedure for the metabolites. Considering the capabilities of the 600 MHz NMR spectrometer used in this study, and under the specific conditions employed, a minimum of 64 transients is necessary to detect metabolites at concentrations as low as 45 μg/mL.
In exploring various pulse sequences that may offer advantages for studying and detecting metabolites in using the above-mentioned model, multiple NMR pulse sequences can be used, each providing unique benefits for the analysis.
Conclusion: This study provided insights about the metabolic changes in HepaRGTM cells induced by caron-13 enriched lactate, pyruvate, and octanoate, providing insights into steatosis progression. By optimizing a protocol involving quenching, cell lysis, and dual phase metabolite extraction, reproducibility and accuracy were ensured. Key metabolites and altered metabolic pathways, such as those related to lipolysis, gluconeogenesis, and the TCA cycle, were identified, offering potential therapeutic targets for steatosis. The integration of 13C-labeled tracers enabled the tracing of carbon-13 within metabolic pathways, revealing detailed metabolic shifts. Despite the study's findings, limitations included sample availability and inherent NMR insensitivity, suggesting the benefit of transitioning to an 800 MHz spectrometer with a cryoprobe for enhanced sensitivity. These findings contribute to a deeper understanding of steatosis and underscore the need for advanced NMR techniques in metabolomics research.
| Date of Award | 24 Jul 2024 |
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| Original language | English |
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| Awarding Institution | - University Of Strathclyde
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| Sponsors | University of Strathclyde |
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| Supervisor | John Parkinson (Supervisor) & Nicholas Rattray (Supervisor) |
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