title: Proteomic profiling of cellular steatosis with concomitant oxidative stress in vitro.
abstract: BACKGROUND
  Nutrient excess underpins the development of nonalcoholic fatty liver disease (NAFLD). The ensuing metabolic derangement is characterised by increased cellular respiration, oxidative stress and mitochondrial impairment. We have previously recapitulated these events in an in vitro cellular steatosis model. Here, we examined the distinct patterns of protein expression involved using a proteomics approach.
  
  
  METHODS
  Human hepatoblastoma C3A cells were treated with a combination of energy substrates; lactate (L), pyruvate (P), octanoate (O) and ammonia (N). Proteins extracts were trypsinized and analyzed on a capillary HPLC OrbitrapXL mass spectrometer. Proteins were quantified using a label-free intensity based approach. Functional enrichment analysis was performed using ToppCluster via Gene Ontology (GO) database.
  
  
  RESULTS
  Of the 1327 proteins identified, 104 were differentially expressed between LPON and untreated cells (defined as: ≥2 peptides; fold change ≥1.5; p-value <0.05). Seventy of these were upregulated with LPON. Functional enrichment analysis revealed enhanced protein biosynthesis accompanied by downregulation of histones H2A type 1-A, H1.2, H1.5 and H1.0I in LPON cells. Lipid binding annotations were also enriched as well as proteins involved in cholesterol synthesis, uptake and efflux. Increased expression of aldo-keto reductase family 1, member C1 and C3 suggests enhanced sterol metabolism and increased ROS-mediated lipid peroxidation.
  
  
  CONCLUSIONS
  The surge of energy substrates diverts free fatty acid metabolism towards pathways that can mitigate lipotoxicity. The histones depletion may represent an adaptation to increased protein synthesis. However, this can also expose DNA to oxidative stress thus should be explored further in the context of NAFLD progression.
authors.affiliation: Hepatology Laboratory, University of Edinburgh, 49 Little France Crescent, Edinburgh, EH16 4SB, Scotland, UK.
authors.forename: Khalida Ann
authors.initials: KA
authors.lastname: Lockman
authors.affiliation: Hepatology Laboratory, University of Edinburgh, 49 Little France Crescent, Edinburgh, EH16 4SB, Scotland, UK.
authors.forename: Varanand
authors.initials: V
authors.lastname: Htun
authors.affiliation: Hepatology Laboratory, University of Edinburgh, 49 Little France Crescent, Edinburgh, EH16 4SB, Scotland, UK.
authors.forename: Rohit
authors.initials: R
authors.lastname: Sinha
authors.affiliation: Hepatology Laboratory, University of Edinburgh, 49 Little France Crescent, Edinburgh, EH16 4SB, Scotland, UK.
authors.forename: Philipp
authors.initials: P
authors.lastname: Treskes
authors.affiliation: Hepatology Laboratory, University of Edinburgh, 49 Little France Crescent, Edinburgh, EH16 4SB, Scotland, UK.
authors.forename: Leonard J
authors.initials: LJ
authors.lastname: Nelson
authors.affiliation: Kinetic Parameter Facility, SynthSys - Centre for Synthetic and Systems Biology, University of Edinburgh, Edinburgh, EH9 3BF, UK.
authors.forename: Sarah F
authors.initials: SF
authors.lastname: Martin
authors.affiliation: Kinetic Parameter Facility, SynthSys - Centre for Synthetic and Systems Biology, University of Edinburgh, Edinburgh, EH9 3BF, UK.
authors.forename: Sophie M
authors.initials: SM
authors.lastname: Rogers
authors.affiliation: Kinetic Parameter Facility, SynthSys - Centre for Synthetic and Systems Biology, University of Edinburgh, Edinburgh, EH9 3BF, UK.
authors.forename: Thierry
authors.initials: T
authors.lastname: Le Bihan
authors.affiliation: Hepatology Laboratory, University of Edinburgh, 49 Little France Crescent, Edinburgh, EH16 4SB, Scotland, UK.
authors.forename: Peter C
authors.initials: PC
authors.lastname: Hayes
authors.affiliation: Hepatology Laboratory, University of Edinburgh, 49 Little France Crescent, Edinburgh, EH16 4SB, Scotland, UK. j.plevris@ed.ac.uk.
authors.forename: John N
authors.initials: JN
authors.lastname: Plevris
mesh_terms: D000449:Aldehyde Reductase / Q000235:genetics / Q000378:metabolism; D000074408:Aldo-Keto Reductases; D000641:Ammonia / Q000494:pharmacology*; D002210:Caprylates / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002784:Cholesterol / Q000096:biosynthesis; D005230:Fatty Acids, Nonesterified / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D063990:Gene Ontology; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019344:Lactic Acid / Q000494:pharmacology*; D015227:Lipid Peroxidation; D050155:Lipogenesis / Q000187:drug effects / Q000235:genetics; D008954:Models, Biological; D058977:Molecular Sequence Annotation; D065626:Non-alcoholic Fatty Liver Disease / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D018384:Oxidative Stress; D014176:Protein Biosynthesis / Q000187:drug effects; D040901:Proteomics; D019289:Pyruvic Acid / Q000494:pharmacology*
chemical_list: D002210:Caprylates; D005230:Fatty Acids, Nonesterified; D006657:Histones; D019344:Lactic Acid; D000641:Ammonia; D019289:Pyruvic Acid; D002784:Cholesterol; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase; C031492:octanoic acid
keywords: Nonalcoholic fatty liver; Oxidative stress; Steatohepatitis