Identification of novel biomarkers and treatments for non-alcoholic fatty liver disease and associated co-morbidities
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2022
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Melbourne Authors
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Anatomy and Neuroscience
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PhD thesis
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Open Access
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© 2022 William De Nardo
Abstract
Non-alcoholic fatty liver disease (NAFLD) is characterised by impaired lipid metabolism, hepatic fibrosis and linked to systemic insulin resistance, which is partly mediated by remodelling of liver-secreted proteins or “hepatokines.” There are no effective screening tools or approved pharmacotherapies for NAFLD and NAFLD-associated fibrosis. Proteomic studies estimate that ~25% of circulating proteins are liver-derived, suggesting that hepatokines could be biomarkers and, due to well described impacts on energy homeostasis, effective treatments for NAFLD and NAFLD-associated fibrosis.
This thesis aimed to discover novel biomarkers and treatments for NAFLD, non-alcoholic steatohepatitis (NASH), and NAFLD with significant fibrosis (NAFLD F2-3). For biomarker discovery, bariatric patients were prospectively recruited, plasma and a liver wedge biopsy were procured, livers were precision-cut to assess hepatokine secretion and were grouped based on histological confirmation of NAFLD, NASH, and NAFLD F2-3. Chapter 3 identified 3333 liver-secreted proteins, of which, 107 and 5 hepatokines were remodelled with NASH and NAFLD F2-3, respectively. EMILIN1 secretion was increased in NASH, NAFLD F2-3, and in the plasma of NASH patients. However, plasma EMILIN1 poorly stratified patients NASH. In Chapter 4, we speculated that hepatokine secretion may not correspond to changes in plasma levels. Notably, highly abundant plasma proteins corresponded to ~62% of the proteins detected in the human liver-secreted proteome. Plasma proteomics showed few changes in patients with NASH and NAFLD F2-3. Proteins APOF and FCN3 stratified patients with NASH and IGKV2-28 and APCS outcompeted existing non-invasive scores to stratify patients with NAFLD F2-3. Together, this provides a novel resource characterising human liver-secreted and highly abundant plasma proteins and identified IGKV2-28, APCS, APOF, and FCN3 as biomarkers for NAFLD-associated fibrosis and NASH.
In Chapters 5 and 6, we exploited hepatokine remodelling to uncover novel treatments for NAFLD and hepatic fibrosis. In Chapter 3, we detected EMILIN1, which was previously reported to reduce TGF signalling. Hepatocyte-specific overexpression of EMILIN1 with severe NASH and advanced fibrosis reduced TGF signalling and hepatic fibrosis, while overexpression of EMILIN1 mice in a mouse model of mild NASH did not impact fibrosis or metabolic co-morbidities, suggesting that EMILIN1 could be a therapeutic target for advanced, but not mild, fibrosis.
Physical activity can resolve NAFLD and improve its metabolic co-morbidities, however, the effects of exercise-training on hepatokine secretion and the metabolic impact of exercise-regulated hepatokines in NAFLD remain unresolved. Chapter 6 assessed whether exercise-training could remodel hepatokine secretion to mediate improvements in NAFLD and associated co-morbidities. Mass spectrometry proteomics analysis detected 2657 intracellular and 1593 secreted proteins from isolated mouse hepatocytes. Exercise-training remodelled the hepatocyte proteome, with differences in 137 intracellular and 35 secreted proteins. Hepatocyte-secreted factors from exercise-trained mice improved insulin action in skeletal muscle and increased hepatic fatty acid oxidation. Hepatocyte-specific overexpression of SDC4 reduced hepatic steatosis, which was associated with reduced fatty acid uptake, and blunted pro-inflammatory and pro-fibrotic gene expression. Treating hepatocytes with recombinant SDC4 recapitulated these effects.
Together, the work in this thesis addresses an unmet clinical need to discover novel biomarkers and therapeutics to treat NAFLD and co-morbidities.
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NAFLD, NASH, fibrosis, Exercise, Proteomics, Hepatokines, EMILIN1, Syndecan 4, Biomarkers