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

Santrampurwala, Nishreen (2016). Hfe-associated steatohepatitis: expression profiling and identifying the molecular basis of liver injury PhD Thesis, School of Medicine, The University of Queensland. doi:10.14264/uql.2016.365

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Author Santrampurwala, Nishreen
Thesis Title Hfe-associated steatohepatitis: expression profiling and identifying the molecular basis of liver injury
Formatted title
Hfe-associated steatohepatitis: expression profiling and identifying the molecular basis of liver injury
School, Centre or Institute School of Medicine
Institution The University of Queensland
DOI 10.14264/uql.2016.365
Publication date 2016-06-20
Thesis type PhD Thesis
Supervisor Darrell Crawford
Nathan Subramaniam
Kim Bridle
Total pages 207
Total colour pages 33
Total black and white pages 174
Language eng
Subjects 1103 Clinical Sciences
Formatted 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. Non-alcoholic 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 iron-induced 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 in Hfe-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 cytokine, Ccl5, indicative of a more severe injury phenotype with co-administration of FFA and iron. This model was also used to investigate hepcidin (Hamp1) expression, a key regulator II of systemic iron homeostasis, in the setting of lipid accumulation. Despite iron loading, mice fed a HCD had markedly reduced expression of Hamp1. BMP (bone morphogenetic protein)-SMAD signalling is known to be important in hepcidin induction therefore it was hypothesised that BMP signalling is altered in a fatty acid-rich environment. Consistent with this hypothesis, this study found reduced expression of BMP6 target genes when stimulated with exogenous BMP6 in cells treated with free fatty acids (FFA). This blunted response to BMP6 stimulation was due to the reduced activation of SMAD1/5/8 which is an essential component of the BMP-SMAD signalling cascade.

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 hepatocytes and it was hypothesised that the reduced expression of Arsg, which causes lysosomal storage pathology in the brains, may indicate the role for lysosomal pathology in the development of steatohepatitis. Lastly Ifi27l2b, contrary to its described role as an interferon stimulated gene, did not cause increased apoptosis and its expression was positively correlated with protein kinase B (pAKT), a crucial protein in insulin signalling.

Kupffer cell iron loading is a common occurrence in NASH therefore the expression of ARSG, GPLD1 and IFI27L2B in iron-loaded and LPS-activated macrophages was also examined in this study. Iron administration did not result in activation of an inflammatory response in the macrophages and also did not alter the expression of ARSG, GPLD1 and IFI27L2B. However, expression of all three proteins was reduced in LPS-activated macrophages. This warrants further analysis of these genes in macrophages and the subsequent effects on hepatocyte phenotype.

In summary, studies in this thesis have identified 3 genes involved in NASH pathogenesis and have outlined their expression with a variety of external stimuli associated with the development of NASH. These studies lay the foundation for future work in this area with a particular interest in the co-administration of FFA and iron in driving changes in gene expression.
Keyword Hfe-/- mice
High calorie diet
Free fatty acids

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Created: Wed, 01 Jun 2016, 17:05:09 EST by Nishreen Santrampurwala on behalf of Learning and Research Services (UQ Library)