Non-Alcoholic Fatty Liver Disease : Insights into Alcohol Consumption, Genetics, and Proteomics

Abstract: NAFLD (Non-Alcoholic Fatty Liver Disease) affects approximately a quarter of the global population and is closely linked to type 2 diabetes mellitus and obesity. The disease spectrum ranges from steatosis and steatohepatitis to fibrosis, cirrhosis, and hepatocellular cancer. However, accurately predicting which patients will experience a progressive disease course remains a significant challenge. The variant gene of PNPLA3 is known to be associated with NAFLD and a more progressive disease, although its precise function remains unclear.   Patients with NAFLD typically consume small to moderate amounts of alcohol, with recommended thresholds set at a maximum of 210 gram per week for males and 140 grams per week in females. However, the impact of alcohol consumption on liver disease in NAFLD remains disputed, with conflicting research findings.   Liver biopsy is considered the gold standard for diagnosing NAFLD. However, due to its impracticality for such a large population with the condition, various non-invasive methods have been explored for diagnosing and evaluating NAFLD.  This thesis aimed to investigate the potential effects of moderate alcohol consumption on NAFLD histology, explore the potential role of variant PNPLA3 in NAFLD, and assess the use of proteomics in classifying fibrosis.  In Papers I and II, moderate alcohol consumption was assessed through questionnaires, clinical interviews, and measurement of the direct alcohol biomarker phosphatidylethanol (PEth). Paper I, a cross-sectional study including 86 participants, showed an association between moderate consumption and advanced fibrosis. Moderate consumption was defined as consuming more than 66 grams of ethanol per week or a PEth-value over 50 ng/mL. Notably, individuals with both moderate alcohol consumption and a diagnosis of type 2 diabetes exhibited significantly more advanced fibrosis. Paper II was a cohort study where 82 participants were followed over 17.2 years. Similarly, participants with moderate alcohol consumption displayed significant fibrosis progression. The strongest association was observed in participants with PEth-value of 48 ng/mL or higher, or those with binge drinking.  In Paper III, the potential role of variant PNPLA3 was explored, exhibiting impaired autophagic flux and reduced lipophagy in variant PNPLA3 cells. Liver biopsies of NAFLD individuals with variant PNPLA3 displayed an accumulation of lipid droplets positive for both PNPLA3 and LC3 (a common marker of the autophagosome). This suggests that PNPLA3 is part of the lipophagy process, which is impaired in the variant gene and contributes to steatosis.  Paper IV examined two independent NAFLD cohorts. In the discovery cohort, 60 participants with biopsy-proven NAFLD were included, while 59 participants were included in the validation cohort. The study evaluated 266 proteins and found that a biomarker model combining ACE2, HGF, and IGFBP-7 distinguished between different fibrosis stages (F0–1 and F2–4) in both cohorts.  In summary, measuring phosphatidylethanol is advisable in NAFLD patient evaluations. Elevated PEth-levels (≥48 ng/mL) or alcohol consumption exceeding 66 grams per week should warrant advice to abstain from alcohol. PNPLA3 is implicated in NAFLD pathophysiology, potentially through impaired lipophagy. While its clinical application remains uncertain, genetic profiling for NAFLD risk assessment may become part of future non-invasive approaches. Additionally, proteomics holds promise for non-invasive NAFLD assessment, with the combination of ACE2, HGF, and IGFBP-7 identifying significant fibrosis in two separate cohorts.