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The mechanism of how patatin-like phospholipase domain-containing protein 3 (PNPLA3) variant M148 is associated with increased risk of development of hepatic steatosis is still debated. Here, we propose a novel role of PNPLA3 as a key player during autophagosome formation in the process of lipophagy. A human hepatocyte cell line, HepG2 cells, expressing recombinant I148 or 148M, was used to study lipophagy under energy deprived conditions, and lipid droplet morphology was investigated using florescence microscopy, image analysis and biochemical assays. Autophagic flux was studied using the golden-standard of LC3-II turnover in combination with the well characterized GFP-RFP-LC3 vector. To discriminate between, perturbed autophagic initiation and lysosome functionality, lysosomes were characterized by Lysotracker staining and LAMP1 protein levels as well as activity and activation of cathepsin B. For validation, human liver biopsies genotyped for I148 and 148M were analyzed for the presence of LC3-II and PNPLA3 on lipid droplets. We show that the M148-PNPLA3 variant is associated with lipid droplets that are resistant to starvation-mediated degradation. M148 expressing hepatocytes reveal decreased autophagic flux and reduced lipophagy. Both I148-PNPLA3 and M148-PNPLA3 colocalize and interact with LC3-II, but the M148-PNPLA3 variant has lower ability to bind LC3-II. Together, our data indicate that PNPLA3 might play an essential role in lipophagy in hepatocytes and furthermore that the M148-PNPLA3 variant appears to display a loss in this activity, leading to decreased lipophagy.
Non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease affecting approximately 25% of the global population. Although a majority of NAFLD patients will never experience liver-related symptoms it is estimated that 5-10% will develop cirrhosisrelated complications with risk of death or need for liver transplantation. NAFLD is closely associated with cardiovascular disease and components of the metabolic syndrome. However, NAFLD is not uncommon in lean individuals and may in these subjects represent a different entity with separate pathophysiological mechanisms involved implying a higher risk for development of end-stage liver disease. There is considerable fluctuation in the histopathological course of NAFLD that may partly be attributed to lifestyle factors and dietary composition. Nutrients such as fructose, monounsaturated fatty acids, and trans-fatty acids may aggravate NAFLD. Presence of type 2 diabetes mellitus seems to be the most important clinical predictor of liver-related morbidity and mortality in NAFLD. Apart from severity of the metabolic syndrome, genetic polymorphisms and environmental factors, such as moderate alcohol consumption, may explain the variation in histopathological and clinical outcome among NAFLD patients.
The effect of moderate alcohol consumption on NAFLD histology is disputed. Assessment of alcohol consumption is commonly performed with interview or questionnaires. Phosphatidylethanol (PEth) in blood is a highly sensitive and specific alcohol biomarker, which only forms in the presence of ethanol. PEth has hitherto not been evaluated in longitudinal NAFLD studies. This study aimed to examine the impact of moderate alcohol consumption on histologic progression and evaluate the utility of PEth in NAFLD. NAFLD patients with serial biopsies were reviewed for inclusion in the study. At baseline, all patients reported alcohol consumption <140 g/week. Anthropometric and biochemical measurements were performed at baseline and follow-up. Alcohol consumption was assessed thoroughly at follow-up with clinical interview, the Alcohol Use Disorder Identification Test-Consumption (AUDIT-C) questionnaire, and analysis of PEth in whole blood. Eighty-two patients were included. Mean follow-up time was 17.2 years (SD±6.0). Patients with significant fibrosis progression (defined as progression of ≥2 stages or development of cirrhosis-related complications) reported higher alcohol consumption and had significantly higher PEth. Consumption >66–96 g/week (but <140 g) (i.e. moderate alcohol consumption) was associated with increased risk of significant fibrosis progression compared with no or low consumption. PEth ≥48 ng/mL and binge drinking showed the highest risk for significant fibrosis progression (aOR: 5.9; 95% CI: 1.6–21.4) and aOR: 5.1; 95% CI: 1.4–18.1, respectively). NAFLD patients consuming moderate amounts of alcohol are at increased risk for significant fibrosis progression and development of cirrhosis-related complications. PEth is a potential biomarker to assess harmful alcohol consumption in NAFLD. Patients reporting moderate consumption or exhibiting PEth ≥48 ng/mL should be advised to reduce alcohol consumption.
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