Nonalcoholic fatty liver disease (NAFLD) is a clinicopathological condition which results in hepatic damage associated with significant lipid accumulation in the liver of patients without a history of excessive alcohol use (Mulhall et al. 2002). NAFLD is a clinical condition that refers to a spectmm of liver abnormalities from fatty liver, nonalcoholic steatohepatitis (NASH) and eventually cirrhosis (Yngve et al. 2001 and Matteoni et al. 1999), and is becoming increasingly recognized as a common and potentially serious form of liver disease that may progress to end-stage liver disease (Yu and Keeffe et al. 2002).
The work described in this thesis had two principal aims. The fust was to quantify differences in gene expression between normal donor livers, fatty livers and livers from patients with NASH, to validate existing DNA microarray data. The second was to determine whether de novo lipogenesis plays a role in the pathogenesis of NAFLD.
Analysis and validation of gene expression
DNA microarray analyses have previously shown that expression of a subset of genes was significantly differentially expressed in liver subjects with NAFLD (Gochee 2002). In chapter three of this thesis, a panel of six genes, encoding plasma proteins, was chosen as targets for analysis. Relative mRNA expression levels of phospholipase A2 (PLA2G2A); chitinase-3 like protein 1(CHI3L1); selenoprotein P (SELP); alpha-2-HS-glycoprotein (AHSG); plasma glutathione peroxidase (GPX3); and ceruloplasmin (CP) were determined by real-time PCR, using cDNA prepared from liver biopsies from subjects with NASH. The mRNA expression levels in biopsies were compared with expression levels in donor livers from adults with steatosis and in donor livers from adults previously recorded as being non-diseased. The results demonstrate that expression of the genes PLA2G2A, CHI3L1, and CP was significantly down-regulated in NASH livers relative to non-diseased donor livers. These observations are in agreement with the DNA microarray analysis of paediatric NASH livers. Expression of the gene AHSG was significantly up-regulated in adult NASH livers, relative to non-diseased controls livers. These results also confirmed the analysis of paediatric NASH livers using DNA microarrays. However, expressions of SELP and of GPX3 were not significant.
We also analysed gene expression levels in steatotic donor livers. The results of these studies showed that only expression of PLA2G2A differed between steatotic donor livers and control donor livers. The results in chapter 3 also show that the expressions of some genes are highly correlated with each other.
Taken together these results demonstrate that the expression levels of a subgroup of the genes analysed are elevated or decreased in both paediatric and adult livers. The corresponding proteins may prove to be useful as serum biomarkers for NASH. However, the fact that the expression of some of the genes is correlated may indicate that these proteins are not all independent biomarkers.
Role of De novo lipogenesis in the pathogenesis of NAFLD
Steatosis is a common feature of the different stages of NAFLD. The accumulation of triglycerides (TG) in hepatocytes is caused by a disturbed balance between lipogenesis, lipolysis/fatty acid oxidation and the secretion of TG in very low density lipoproteins. The conventional explanation for this imbalance is that obesity and insulin resistance leads to (i) increased hydrolysis of TG in adipocytes, (ii) a concomitant release of free fatty acids (FFAs) that are taken up by the liver in a largely unregulated manner, and (iii) re-esterification of FFAs to form TGs.
The aim of the present study was to determine the role of de novo fatty acid synthesis to NAFLD. To this end, we used real-time PCR to measure relative mRNA levels for lipogenic genes and for genes of the SREBP pathway in liver biopsies from subjects with NASH, and in non-diseased donor livers. The data show that expression of the fatty acid synthase (FAS) gene was more than 11- fold higher in NASH livers than in control livers, consistent with the view that fatty acid synthesis contributes significantly to steatosis in NASH livers. Sterol regulatory element binding protein (SREBP)-1c is known to be activated by insulin in the liver, leading to transcriptional activation of genes required for fatty acid synthesis. Surprisingly, neither SREBP-1c, nor the other isoforms were differentially expressed in NAFLD livers. The two endoplasmic reticulum retention proteins lnsig-1 and Insig-2 were also expressed at similar levels in NASH and control livers. However, transcripts for the escort protein SCAP were more than 3-fold higher in NASH livers than in control livers. Furthermore, FAS mRNA levels were correlated with SCAP mRNA levels. We propose a model in which an increased SCAP concentration in the ER alters the balance between SCAP and its retention proteins Insig-1 and -2, leading to enhanced transfer of the SREBP/SCAP complex from the ER to the Golgi, where SREBP is processed to the transcriptionally active form that activates transcription of the FAS gene.