title: Oxidative stress rather than triglyceride accumulation is a determinant of mitochondrial dysfunction in in vitro models of hepatic cellular steatosis.
abstract: OBJECTIVE
  There is still debate about the relationship between fat accumulation and mitochondrial function in nonalcoholic fatty liver disease. It is a critical question as only a small proportion of individuals with steatosis progress to steatohepatitis. In this study, we focused on defining (i) the effects of triglyceride accumulation and reactive oxygen species (ROS) on mitochondrial function (ii) the contributions of triglyceride, ROS and subsequent mitochondrial impairment on the metabolism of energy substrates.
  
  
  METHODS
  Human hepatoblastoma C3A cells, were treated with various combinations of oleate, octanoate, lactate (L), pyruvate (P) and ammonia (N) acutely or for 72 h, before measurements of triglyceride concentration, cell respiration, ROS production, mitochondrial membrane potential, ketogenesis and gluconeogenesis, TCA cycle metabolite analysis and electron microscopy.
  
  
  RESULTS
  Acutely, LPON treatment enhanced mitochondrial respiration and ROS formation. After 72 h, despite the similarities in triglyceride accumulation, LPON treatment, but not oleate, dramatically affected mitochondrial function as evidenced by decreased respiration, increased mitochondrial membrane potential and ROS formation with concomitant enhanced ketogenesis. By comparison, respiration and ROS formation remained unperturbed with oleate. Importantly, this was accompanied by an increased gluconeogenesis and ketogenesis. The addition of the antioxidant N-acetyl-L-cysteine prevented mitochondrial dysfunction and reversed metabolic changes seen with LPON, strongly suggesting ROS involvement in mediating mitochondrial impairment.
  
  
  CONCLUSIONS
  Our data indicate that ROS formation, rather than cellular steatosis per se, impairs mitochondrial function. Thus, reduction in cellular steatosis may not always be the desired outcome without concomitant improvement in mitochondrial function and/or reducing of ROS formation.
authors.affiliation: Department of Diabetes and Endocrinology, University of Edinburgh and Royal Infirmary of Edinburgh, Edinburgh, UK. k.a.lockman@sms.ed.ac.uk
authors.forename: Khalida A
authors.initials: KA
authors.lastname: Lockman
authors.forename: James P
authors.initials: JP
authors.lastname: Baren
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Pemberton
authors.forename: Hussam
authors.initials: H
authors.lastname: Baghdadi
authors.forename: Karl E
authors.initials: KE
authors.lastname: Burgess
authors.forename: Nikolas
authors.initials: N
authors.lastname: Plevris-Papaioannou
authors.forename: Patricia
authors.initials: P
authors.lastname: Lee
authors.forename: Forbes
authors.initials: F
authors.lastname: Howie
authors.forename: Geoff
authors.initials: G
authors.lastname: Beckett
authors.forename: Anne
authors.initials: A
authors.lastname: Pryde
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Jaap
authors.forename: Peter C
authors.initials: PC
authors.lastname: Hayes
authors.forename: Celine
authors.initials: C
authors.lastname: Filippi
authors.forename: John N
authors.initials: JN
authors.lastname: Plevris
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology; D045744:Cell Line, Tumor; D019069:Cell Respiration; D005234:Fatty Liver / Q000378:metabolism*; D005943:Gluconeogenesis / Q000187:drug effects / Q000502:physiology; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects / Q000502:physiology; D008930:Mitochondria, Liver / Q000378:metabolism* / Q000648:ultrastructure; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000378:metabolism; D014280:Triglycerides / Q000032:analysis / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D000975:Antioxidants; D017382:Reactive Oxygen Species; D014280:Triglycerides; D000111:Acetylcysteine