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Non-Alcoholic Fatty Liver Disease: Insights into Alcohol Consumption, Genetics, and Proteomics
Linköping University, Department of Health, Medicine and Caring Sciences, Division of Diagnostics and Specialist Medicine. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Surgery, Orthopaedics and Cancer Treatment, Mag- tarmmedicinska kliniken.ORCID iD: 0000-0002-6364-8758
2023 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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. 

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2023. , p. 82
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1866
National Category
Gastroenterology and Hepatology
Identifiers
URN: urn:nbn:se:liu:diva-198651DOI: 10.3384/9789180752923ISBN: 9789180752916 (print)ISBN: 9789180752923 (electronic)OAI: oai:DiVA.org:liu-198651DiVA, id: diva2:1806581
Public defence
2023-11-24, Berzeliussalen, building 463, Campus US, Linköping, 13:00 (English)
Opponent
Supervisors
Available from: 2023-10-23 Created: 2023-10-23 Last updated: 2023-10-23Bibliographically approved
List of papers
1. Moderate alcohol consumption is associated with advanced fibrosis in non-alcoholic fatty liver disease and shows a synergistic effect with type 2 diabetes mellitus
Open this publication in new window or tab >>Moderate alcohol consumption is associated with advanced fibrosis in non-alcoholic fatty liver disease and shows a synergistic effect with type 2 diabetes mellitus
2021 (English)In: Metabolism: Clinical and Experimental, ISSN 0026-0495, E-ISSN 1532-8600, Vol. 115, article id 154439Article in journal (Refereed) Published
Abstract [en]

Background: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide. Whether moderate alcohol consumption plays a role for progression of NAFLD is disputed. Moreover, it is not known which tool is ideal for assessment of alcohol consumption in NAFLD. This study aimed to evaluate if moderate alcohol consumption assessed with different methods, including the biological marker phosphatidylethanol (PEth), is associated with advanced fibrosis in NAFLD. Methods: We conducted a cross-sectional study of patients with biopsy-proven NAFLD. All participants were clinically evaluated with medical history, blood tests, and anthropometric measurements. Alcohol consumption was assessed using PEth in blood, the questionnaire AUDIT-C, and clinical interview. Findings: 86 patients were included of which 17% had advanced fibrosis. All participants reported alcohol consumption < 140 g/week. Average weekly alcohol consumption was higher in the group with advanced fibrosis. Moderate alcohol consumption, independently of the method of assessment, was associated with increased probability of advanced fibrosis (adjusted OR 5.5-9.7, 95% CI 1.05-69.6). Patients with type 2 diabetes mellitus (T2DM) consuming moderate amounts of alcohol had a significantly higher rate of advanced fibrosis compared with those consuming low amounts (50.0-60.0% vs. 33-21.6%, p < 0.05). Conclusions: Moderate alcohol consumption, irrespective of assessment method (clinical interview, AUDIT-C, and PEth), was associated with advanced fibrosis. PEth in blood >= 50 ng/mL may be a biological marker indicating increased risk for advanced fibrosis in NAFLD. Patients with T2DM consuming moderate amounts of alcohol had the highest risk of advanced fibrosis, indicating a synergistic effect of insulin resistance and alcohol on the histopathological progression of NAFLD. (C) 2020 The Author(s). Published by Elsevier Inc.

Place, publisher, year, edition, pages
Elsevier, 2021
Keywords
Non-alcoholic fatty liver disease; Alcohol drinking; Type 2 diabetes mellitus; Phosphatidylethanol
National Category
Surgery
Identifiers
urn:nbn:se:liu:diva-173399 (URN)10.1016/j.metabol.2020.154439 (DOI)000608790700005 ()33246008 (PubMedID)2-s2.0-85097192291 (Scopus ID)
Note

Funding Agencies|Medical Research Council of Southeast SwedenUK Research & Innovation (UKRI)Medical Research Council UK (MRC)

Available from: 2021-02-20 Created: 2021-02-20 Last updated: 2023-10-23Bibliographically approved
2. Moderate alcohol consumption is associated with significant fibrosis progression in NAFLD
Open this publication in new window or tab >>Moderate alcohol consumption is associated with significant fibrosis progression in NAFLD
2023 (English)In: HEPATOLOGY COMMUNICATIONS, ISSN 2471-254X, Vol. 7, no 1Article in journal (Refereed) Published
Abstract [en]

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 & PLUSMN;6.0). Patients with significant fibrosis progression (defined as progression of & GE;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 & GE;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 & GE;48 ng/mL should be advised to reduce alcohol consumption.

Place, publisher, year, edition, pages
LIPPINCOTT WILLIAMS & WILKINS, 2023
National Category
Substance Abuse
Identifiers
urn:nbn:se:liu:diva-197483 (URN)10.1097/HC9.0000000000000003 (DOI)001038261400003 ()36633482 (PubMedID)
Note

Funding Agencies|~ALF Grants; Region Ostergoetland; Medical Research Council of Southeast Sweden [752871]

Available from: 2023-09-12 Created: 2023-09-12 Last updated: 2024-02-08
3. PNPLA3 variant M148 causes resistance to starvation-mediated lipid droplet autophagy in human hepatocytes
Open this publication in new window or tab >>PNPLA3 variant M148 causes resistance to starvation-mediated lipid droplet autophagy in human hepatocytes
Show others...
2019 (English)In: Journal of Cellular Biochemistry, ISSN 0730-2312, E-ISSN 1097-4644, Vol. 120, no 1, p. 343-356Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
John Wiley & Sons, 2019
Keywords
adiponutrin; lipophagy; liver; steatosis
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:liu:diva-153347 (URN)10.1002/jcb.27378 (DOI)000450823500032 ()30171718 (PubMedID)2-s2.0-85052837166 (Scopus ID)
Note

Funding Agencies|Swedish Research Foundation [11284]; Crafoord Foundation; Swedish Diabetes Foundation; Albert Pahlsson Foundation; Dr Per Hakansson Foundation

Available from: 2018-12-13 Created: 2018-12-13 Last updated: 2023-10-23Bibliographically approved

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Blomdahl, Julia

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