| dc.description.abstract | Non-alcoholic fatty liver disease (NAFLD) is the most common type of chronic liver disease in the Western countries. NAFLD encompasses the entire spectrum of fatty liver disease in individuals without significant alcohol consumption, ranging from nonalcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH) and cirrhosis. The molecular events that influence disease progression from NAFL to aggressive NASH remain poorly understood; leading to a lack of mechanism-based targeted treatment options for NASH. Therefore, an increased understanding of NASH pathogenesis is pertinent to improve disease interventions in the future. The first objective of this dissertation was to identify key signatures associated with disease progression from NAFL to NASH. Using a diet high in fructose, cholesterol, and fat (HFCF) for up to 9 months, we were able to induce NAFLD spectrum similar to humans in C57BL/6J mice. Specifically, the mice sequentially developed steatosis, steatohepatitis, steatohepatitis with fibrosis, and eventually spontaneous liver tumor as time on the HFCF diet progressed. These data indicate this NAFLD mouse model with disease progression from NAFL to NASH recapitulates key metabolic, and histologic changes seen in humans and could be used for further studies. It is well known that NASH is characterized by steatosis plus inflammation with or without fibrosis. Nuclear receptor, small heterodimer partner (SHP, NR0B2), plays a complex role in lipid metabolism and inflammation. The expression of SHP is negatively correlated with NAFLD progression, indicative of a biological relevance of SHP in NAFLD. Therefore, the second objective of this dissertation was to understand the mechanism behind SHP suppression in NAFLD. Using palmitic acid (PA) or lipopolysaccharide (LPS) combined with pathway inhibitors, we observed that only the inhibition of c-Jun N-terminal kinase (JNK) activation rescued Shp expression. Mechanistically, we found that the downstream target of JNK, c-JUN, directly binds to the promoter region of Shp, preventing activation of Shp by liver receptor homolog-1. The next objective of this dissertation was to understand the role of hepatic SHP in NAFLD. By treating mice that lack or overexpress hepatic SHP with normal chow or HFCF diet, we sought to determine SHP’s role in regulating steatosis and inflammation. We uncovered that hepatocyte Shp deletion (ShpHep-/-) in mice resulted in increased chemokine (C-C motif) ligand 2 (CCL2) secretion which led to a massive infiltration of macrophages and CD4+ T cells to the liver. Additionally, ShpHep-/- mice developed reduced steatosis, but surprisingly increased hepatic inflammation and fibrosis after being fed the HFCF diet. Supporting this data was the RNA-seq analysis which revealed that pathways involved in inflammation and fibrosis were significantly activated in the livers of ShpHep-/- mice fed a chow diet. After being fed the HFCF diet, wildtype mice displayed up-regulated peroxisome proliferator-activated receptor g (Pparg) signaling in the liver; however, this response was completely abolished in the HFCF-fed ShpHep-/- mice. Additionally, ShpHep-/- mice had consistent hepatic nuclear factor κB (NF-κB) activation. To further characterize the role of Shp specifically in the transition of steatosis to NASH, mice were fed the HFCF diet for 4 weeks followed by Shp deletion. Surprisingly, Shp deletion after steatosis development exacerbated hepatic inflammation and fibrosis without affecting liver steatosis. RNA sequencing of hepatic mRNA from mice on the HFCF for 1 month (NAFL) and 3 months (transitioning to NASH) revealed that the key signatures associated with the progression of steatosis to steatohepatitis were related to extracellular matrix organization and immune responses such as leukocyte aggregation, chemotaxis, phagocytosis, and dendritic cell differentiation. Interestingly, our data also revealed that genes regulated by transcription factor forkhead box M1 (Foxm1) and negative elongation factor complex member E (Nelfe) were significantly altered during the disease progression. This was observed in a mouse NAFLD model as well as in patients with NAFLD. As of now, both regulators have been explored in hepatocellular carcinoma, and studies indicating FOXM1 as a regulator of insulin signaling are surfacing. However, the paucity of studies relating FOXM1 and NELFE to NAFLD development and progression warrants additional investigation. The data presented within this dissertation indicate that the decrease of SHP in NAFLD is due to JNK activation of c-Jun which inhibits Shp transcription. Importantly, we demonstrate that, depending on the stage of NASH, hepatic Shp plays an opposing role in steatosis and inflammation. Mechanistically, Shp deletion in hepatocytes activates NF-κB and impairs Pparg activation, leading to the dissociation of steatosis, inflammation, and fibrosis in NASH development. Lastly, RNA-seq analysis of our mouse NAFL and early-stage NASH models reveals the prospective involvement of transcription factors, FOXM1 and NELFE in NASH development. Overall, this dissertation research provides new insights into understanding the regulation of NAFLD progression from NAFL to NASH with the intention to aid in development of novel preventative, diagnostic, and therapeutic strategies for NASH. | |
