Hfe-associated steatohepatitis: expression profiling and identifying the molecular basis of liver injury

Abstract

Non-alcoholic fatty liver disease (NAFLD) is an increasingly common clinical diagnosis and is predicted to become the leading indication for liver transplantation within a decade. Nonalcoholic steatohepatitis (NASH) is the progressive form of NAFLD with a fibro-inflammatory phenotype and can lead to liver cirrhosis. Although the factors responsible for progression from simple steatosis to NASH are unknown, there has been a particular interest in the role of ironinduced oxidative stress in this transition. The HFE protein plays a vital role in maintaining systemic iron homeostasis and a gene mutation in HFE is causative of haemochromatosis. In HFE-haemochromatosis, steatosis is associated with increased liver fibrosis. In keeping with these results, research from our laboratory has previously shown altered lipid metabolism and greater severity of injury in Hfe -/-mice fed a high calorie diet (HCD), which represents a western diet.The primary aim of this project was to enhance the understanding of disease progression inHfe-associated steatohepatitis through the identification and characterisation of genes that are differentially expressed. A gene expression profile was generated by high throughput sequencing of messenger RNA isolated from livers of Hfe -/-mice fed a chow diet and those fed a HCD. Subsequent bioinformatics analysis revealed a list of genes that were significantly altered in response to HCD-induced steatohepatitis. Among the genes that were upregulated are lipid droplet proteins, Perilipin 2 (Plin2) and Cell death inducing DFFA-like effector c (Cidec) which have been previously associated with the development of liver steatosis.Glycosylphosphatidylinositol phospholipase D1 (Gpld1), a high-density lipoprotein, was decreased in NASH livers and was the focus of this study because of the contrary upregulation observed in patients with NAFLD. Arylsulfatase G (Arsg) and Interferon, alpha-inducible protein 27 like 2B (Ifi27l2b) were identified as genes without a previously recognised role in the development of liver injury or fat accumulation and the work in this thesis has primarily focussed on elucidating the underlying roles of these genes in liver injury.To further investigate these genes an in vitro model of hepatocyte fat and iron loading was developed. This model showed gene expression which indicated increased, mitochondrial β-oxidation and reduced fatty acid storage in cells with concomitant free fatty acid (FFA) and iron loading. These changes were also associated with an increase in the pro-inflammatory In this thesis, Gpld1 expression was consistently reduced in AML12 hepatocytes, with all external stimuli, FFA and iron, insulin and inflammation. This downregulation was similar to its expression in rodent NASH livers from transcriptomics analysis and suggests that Gpld1 may influence the extent of injury in iron related steatohepatitis. To the best of my knowledge this is the first study to describe a role for Arsg in response to lipid droplet accumulation and inflammation in hepatocyte...