Understanding the role of dietary sulforaphane in regulating metabolic pathways using genomic approaches

Bernuzzi, Federico (2021) Understanding the role of dietary sulforaphane in regulating metabolic pathways using genomic approaches. Doctoral thesis, University of East Anglia.

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Introduction: Sulforaphane (4-methyl sulfinylbutyl isothiocyanate), a phytochemical derived from broccoli, has been linked to many health benefits in model systems, primarily through the activation of NRF2 (Nuclear factor erythroid-2 related factor), which regulates cellular antioxidant response. Recent evidence suggests that SF may improve glucose regulation in diabetic patients, but the molecular pathways or the role of NRF2 are yet unclear. This work set out to assess the molecular mechanisms by which SF regulates energy metabolism in the liver under conditions that represent different cellular metabolic states. Methods: Established liver hepatocellular carcinoma cells (HepG2) were treated with physiological concentrations of SF (10 µM) under varying glucose concentrations; no (0 mM), basal (5 mM), and high glucose (25 mM). Metabolic phenotyping was undertaken using the Seahorse Extracellular Flux Analyser, untargeted metabolomics, and subsequent experiments with stable isotope tracers of glucose and glutamine, coupled with Gas-Chromatography and Mass Spectrometry (GC-MS). Whole transcriptome was obtained through Illumina RNA sequencing. Finally, the genome-editing technique CRISPR-Cas 9 was applied to assess whether NRF2 mediates the metabolic changes. Results: Real time energy production assessed using the Seahorse Extracellular Flux Analyser demonstrated that SF reduced both mitochondrial respiration and glycolysis in HepG2 cells in a high glucose environment. At the same time, the expression of GSH biosynthetic genes and levels of reduced glutathione (GSH) were significantly increased. To support GSH synthesis, SF altered levels of the three amino acids that are the biosynthetic building blocks; namely, increased intracellular utilization of glycine and glutamate, by redirecting the latter away from the TCA cycle, as well as increased the import of cysteine from the media. To support the cellular antioxidant enzyme response, SF also altered pathways generating NADPH, the necessary cofactor for these oxidoreductase reactions, namely pentose phosphate pathway (PPP) and 1Cmetabolism. Firstly, SF increased genes in the PPP pathway, including glucose-6- phosphate dehydrogenase, the rate limiting enzyme, and increased the PPP metabolite ribulose-5-phosphate, suggesting that excess glucose is likely redirected towards PPP, away from glycolysis. Secondly, SF upregulated genes in the folate cycle, namely 10- formyltetrahydrofolate dehydrogenase (ALDH1L1) and monofunctional C1- 3 tetrahydrofolate synthase (MTHFD1L) and utilized serine as a methyl donor for THF to support the 1C metabolism. Finally, SF downregulated the biosynthesis of the unsaturated fatty acids gene set, which is an NADPH consuming pathway. Finally, transcriptomic and targeted metabolomics LC-MS analysis of NRF2KD HepG2 cells generated using CRISPR-Cas 9 genome editing revealed that the above metabolic effects are mediated through NRF2. Conclusions: The results suggest that SF rewires central metabolism to suppress the metabolic dysregulation induced by excessive glucose and identify glucose biosynthesis and 1Cmetabolism as key mechanistic pathways.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Jackie Webb
Date Deposited: 28 Apr 2022 14:53
Last Modified: 28 Apr 2022 14:53
URI: https://ueaeprints.uea.ac.uk/id/eprint/84841

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