Metabolic Characterisation of Hepatic Stellate Cell Activation: Altered Branched-Chain Amino-Acid Processing as a Novel, Targetable Dependency

Background: Modern dietary and lifestyle factors have led to a sharp rise in metabolic disorders, which manifests in the liver as an accumulation of lipids in hepatocytes, termed metabolic dysfunction associated steatotic liver disease (MASLD). If unchecked, MASLD often leads to hepatic inflammation and fibrosis in the form of metabolic associated steatohepatitis (MASH) which is now the fastest growing cause of cirrhosis. Despite various longitudinal studies identifying aberrant collagen deposition by hepatic stellate cells (HSCs) as the best indicator of poor outcomes in MASH patients, therapies which directly target these cells remain elusive. During MASH pathogenesis, HSCs respond to chemical and mechanical stimuli to undergo a phenotypic switch from a quiescent, vitamin-A storing cell to a proliferative, extracellular matrix (ECM) remodelling myofibroblast. This dramatic change in cell behaviour suggests a concomitant rewiring of intracellular metabolism, with the literature having previously described an upregulation of various enzymes involved in the catabolism of both glucose and glutamine. Any broader changes to the metabolic network in HSCs which support their activation during MASH are still to be elucidated. It was therefore the aim of this thesis to detail the metabolic characteristics of activated HSCs, identify novel metabolic nodes, and to investigate their effects on the fibrogenic phenotype of HSCs. Methods: Initial characterisation of metabolism in activated HSCs was conducted through meta-analysis of publicly available RNAseq data in primary human HSCs in combination with in vitro measurement of both metabolite abundance and substrate usage in both primary human HSCs and immortalised LX-2 HSCs treated with TGF-β1. Subsequent targeting of novel metabolic nodes was done through pharmacological and genetic manipulation of LX-2 HSCs in vitro with characterisation by targeted metabolomics, gene/protein expression analysis, global proteomics, and various phenotypic assays. Results: Here we detail some novel metabolic responses to TGFβ in activated HSCs, observing divergent catabolic flux from carbon substrates through the TCA cycle with a preferential supply of glutamine derived carbon for the de novo synthesis of aspartate. Subsequently we identify a novel dependency on the cytosolic transamination of branched-chain amino-acids through BCAT1 to support the production of both glutamate and aspartate in activated HSCs. Genetic knock-out of BCAT1 in LX-2 HSCs results in depletion of glutamate and aspartate, accumulation of certain TCA cycle intermediates, broad inhibition of protein translation and processing machinery, as well as perturbed collagen deposition and migratory capacity in vitro. Finally, through pharmacological and genetic targeting of BCKDK, with the intention of increasing BCAA oxidation, we show that the enzyme is impactful for HSC activation. Our results also suggest that the effect of BCKDK inhibition on cell phenotype may be vii mediated through phosphorylation targets outside of the BCAA catabolic pathway and that other mechanisms for regulating BCAA oxidation may be employed by activated HSCs. Conclusion: Cumulatively, this thesis adds to the field’s understanding of how metabolism is rewired during HSC driven liver fibrosis, presents the transamination of a cytosolic pool of BCAAs as an important dependency in this pathogenic phenotype, and reveals a complexity to the regulation of BCAA catabolism that has not been previously described in these cells.