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Abstract
The metabolic syndrome and obesity are currently reaching pandemic dimensions worldwide. The hepatic consequence is an imbalance in fatty acid (FA) homeostasis, which results in hepatic lipid accumulation, a critical characteristic of non-alcoholic fatty liver disease (NAFLD), the most common chronic liver disease. NAFLD is a highly heterogeneous liver disease that ranges from simple steatosis (non-alcoholic fatty liver, NAFL), to non-alcoholic steatohepatitis (NASH), which is associated with inflammation and liver injury, and can ultimately lead to NASH-driven hepatocellular carcinoma (HCC). Pro-inflammatory cytokines, derived from activated immune cells, strongly affect hepatic FA metabolism, and are implicated in NASH and NASH-derived HCC. However, underlying molecular mechanisms and signalling pathways are not known in detail yet.
To investigate the influence of NASH-derived inflammatory cytokines on hepatic FA metabolism and to study molecular mechanisms as well as involved signalling pathways, I established an in vitro NASH model to recapitulate the transition from steatosis to steatohepatitis in mouse and human hepatocytes. I used several in vitro and ex vivo experimental set-ups including fluorescence and radioactive labelling of lipids, to study changes in FA- uptake, de novo synthesis, storage, oxidation, and secretion. In addition, I performed fluorescence-based assays to focus on regulations affecting mitochondrial function, cell viability, proliferation and associated replication stress, and DNA damage. By using CRISPR-Cas based knock out cell models and si-RNA mediated knock downs in hepatocytes, as well as treatment of cells with different inhibitors, antagonists, and agonists, I studied involved inflammatory and metabolic pathways. To disentangle molecular mechanisms induced by FAs and pro-inflammatory cytokines, I performed a multi omics approach including lipidome, transcriptome, proteome, phosphoproteome, and thermal proteome profiling.
I demonstrated that inflammatory cytokines increase FA storage in a NF-ĸB dependent manner by interference with catabolic processes. Exacerbated lipid accumulation in hepatocytes subsequently promoted mitochondrial dysfunction, apoptotic cell death and compensatory proliferation. In addition, inflammatory cytokines induced an inflammatory stress response, replication stress, and DNA damage. Importantly, the exposure to inflammatory cytokines alone and in combination with FAs lead to the downregulation of genes and proteins involved in essential metabolic processes in hepatocytes, not only specific for FA metabolism.
The understanding of the effect of inflammatory cytokines on metabolic dysregulation and transcriptional control of metabolic genes in hepatocytes will help to find possible treatment options for NAFLD / NASH.
Document type: | Dissertation |
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Supervisor: | Angel, Prof. Dr. Peter |
Place of Publication: | Heidelberg |
Date of thesis defense: | 5 October 2020 |
Date Deposited: | 10 Mar 2021 11:51 |
Date: | 2021 |
Faculties / Institutes: | The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences |
DDC-classification: | 570 Life sciences |