title: Elevated free cholesterol in a p62 overexpression model of non-alcoholic steatohepatitis.
abstract: OBJECTIVE
  To characterize how insulin-like growth factor 2 (IGF2) mRNA binding protein p62/IMP2-2 promotes steatohepatitis in the absence of dietary cholesterol.
  
  
  METHODS
  Non-alcoholic steatohepatitis (NASH) was induced in wild-type mice and in mice overexpressing p62 specifically in the liver by feeding the mice a methionine and choline deficient (MCD) diet for either two or four weeks. As a control, animals were fed a methionine and choline supplemented diet. Serum triglycerides, cholesterol, glucose, aspartate aminotransferase and alanine transaminase were determined by standard analytical techniques. Hepatic gene expression was determined by real-time reverse transcription-polymerase chain reaction. Generation of reactive oxygen species in liver tissue was quantified as thiobarbituric acid reactive substances using a photometric assay and malondialdehyde as a standard. Tissue fatty acid profiles and cholesterol levels were analyzed by gas chromatography-mass spectrometry after hydrolysis. Hepatocellular iron accumulation was determined by Prussian blue staining in paraffin-embedded formalin-fixed tissue. Filipin staining on frozen liver tissue was used to quantify hepatic free cholesterol levels. Additionally, nuclear localization of the nuclear factor kappa B (NF-κB) subunit p65 was examined in frozen tissues.
  
  
  RESULTS
  Liver-specific overexpression of the insulin-like growth factor 2 mRNA binding protein 2-2 (IGF2BP2-2/IMP2-2/p62) induces steatosis with regular chow and amplifies NASH-induced fibrosis in the MCD mouse model. Activation of NF-κB and expression of NF-κB target genes suggested an increased inflammatory response in p62 transgenic animals. Analysis of hepatic lipid composition revealed an elevation of monounsaturated fatty acids as well as increased hepatic cholesterol. Moreover, serum cholesterol was significantly elevated in p62 transgenic mice. Dietary cholesterol represents a critical factor for the development of NASH from hepatic steatosis. Filipin staining revealed increased free cholesterol in p62 transgenic livers, which were not diet-derived. The mRNA levels of the rate-limiting enzyme for cholesterol synthesis 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase or HMGCR) were not significantly upregulated, potentially due to increased cholesterol biosynthesis via elevated sterol regulatory element binding transcription factor 2 (SREBF2) gene expression and increased iron deposition in transgenic animals.
  
  
  CONCLUSIONS
  This study provides evidence that p62/IGF2BP2-2 drives the progression of NASH through elevation of hepatic iron deposition and increased production of hepatic free cholesterol.
authors.affiliation: Yvette Simon, Sonja M Kessler, Alexandra K Kiemer, Department of Pharmacy, Pharmaceutical Biology, Saarland University, 66123 Saarbrücken, Germany.
authors.forename: Yvette
authors.initials: Y
authors.lastname: Simon
authors.affiliation: Yvette Simon, Sonja M Kessler, Alexandra K Kiemer, Department of Pharmacy, Pharmaceutical Biology, Saarland University, 66123 Saarbrücken, Germany.
authors.forename: Sonja M
authors.initials: SM
authors.lastname: Kessler
authors.affiliation: Yvette Simon, Sonja M Kessler, Alexandra K Kiemer, Department of Pharmacy, Pharmaceutical Biology, Saarland University, 66123 Saarbrücken, Germany.
authors.forename: Katja
authors.initials: K
authors.lastname: Gemperlein
authors.affiliation: Yvette Simon, Sonja M Kessler, Alexandra K Kiemer, Department of Pharmacy, Pharmaceutical Biology, Saarland University, 66123 Saarbrücken, Germany.
authors.forename: Rainer M
authors.initials: RM
authors.lastname: Bohle
authors.affiliation: Yvette Simon, Sonja M Kessler, Alexandra K Kiemer, Department of Pharmacy, Pharmaceutical Biology, Saarland University, 66123 Saarbrücken, Germany.
authors.forename: Rolf
authors.initials: R
authors.lastname: Müller
authors.affiliation: Yvette Simon, Sonja M Kessler, Alexandra K Kiemer, Department of Pharmacy, Pharmaceutical Biology, Saarland University, 66123 Saarbrücken, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Haybaeck
authors.affiliation: Yvette Simon, Sonja M Kessler, Alexandra K Kiemer, Department of Pharmacy, Pharmaceutical Biology, Saarland University, 66123 Saarbrücken, Germany.
authors.forename: Alexandra K
authors.initials: AK
authors.lastname: Kiemer
mesh_terms: D000818:Animals; D002784:Cholesterol / Q000097:blood*; D002796:Choline Deficiency / Q000150:complications; D004195:Disease Models, Animal; D018450:Disease Progression; D005227:Fatty Acids / Q000378:metabolism; D005260:Female; D018836:Inflammation Mediators / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D015227:Lipid Peroxidation; D008099:Liver / Q000378:metabolism*; D008297:Male; D008715:Methionine / Q000172:deficiency*; D008822:Mice, Transgenic; D065626:Non-alcoholic Fatty Liver Disease / Q000097:blood / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D015854:Up-Regulation
chemical_list: D005227:Fatty Acids; C559768:IGF2BP2 protein, mouse; D018836:Inflammation Mediators; D016601:RNA-Binding Proteins; D002784:Cholesterol; D008715:Methionine; D007501:Iron
keywords: Filipin; Insulin-like growth factor 2 mRNA binding protein 2-2; Iron; Methionine/choline deficient; Non-alcoholic fatty liver disease