title: Palmitate-induced activation of mitochondrial metabolism promotes oxidative stress and apoptosis in H4IIEC3 rat hepatocytes.
abstract: OBJECTIVE
  Hepatic lipotoxicity is characterized by reactive oxygen species (ROS) accumulation, mitochondrial dysfunction, and excessive apoptosis, but the precise sequence of biochemical events leading to oxidative damage and cell death remains unclear. The goal of this study was to delineate the role of mitochondrial metabolism in mediating hepatocyte lipotoxicity.
  
  
  METHODS
  We treated H4IIEC3 rat hepatoma cells with free fatty acids in combination with antioxidants and mitochondrial inhibitors designed to block key events in the progression toward apoptosis. We then applied (13)C metabolic flux analysis (MFA) to quantify mitochondrial pathway alterations associated with these treatments.
  
  
  RESULTS
  Treatment with palmitate alone led to a doubling in oxygen uptake rate and in most mitochondrial fluxes. Supplementing culture media with the antioxidant N-acetyl-cysteine (NAC) reduced ROS accumulation and caspase activation and partially restored cell viability. However, (13)C MFA revealed that treatment with NAC did not normalize palmitate-induced metabolic alterations, indicating that neither elevated ROS nor downstream apoptotic events contributed to mitochondrial activation. To directly limit mitochondrial metabolism, the complex I inhibitor phenformin was added to cells treated with palmitate. Phenformin addition eliminated abnormal ROS accumulation, prevented the appearance of apoptotic markers, and normalized mitochondrial carbon flow. Further studies revealed that glutamine provided the primary fuel for elevated mitochondrial metabolism in the presence of palmitate, rather than fatty acid beta-oxidation, and that glutamine consumption could be reduced through co-treatment with phenformin but not NAC.
  
  
  CONCLUSIONS
  Our results indicate that ROS accumulation in palmitate-treated H4IIEC3 cells occurs downstream of altered mitochondrial oxidative metabolism, which is independent of beta-oxidation and precedes apoptosis initiation.
authors.affiliation: Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN, USA.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Egnatchik
authors.affiliation: Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN, USA.
authors.forename: Alexandra K
authors.initials: AK
authors.lastname: Leamy
authors.affiliation: Institute for Innovation, Ajinomoto Co., Inc., Kawasaki, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Noguchi
authors.affiliation: Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, USA.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Shiota
authors.affiliation: Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN, USA; Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, USA. Electronic address: j.d.young@vanderbilt.edu.
authors.forename: Jamey D
authors.initials: JD
authors.lastname: Young
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology*; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D002247:Carbon Isotopes; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D053470:Caspases, Effector / Q000187:drug effects / Q000378:metabolism; D045744:Cell Line, Tumor; D004789:Enzyme Activation / Q000187:drug effects; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism*; D008113:Liver Neoplasms / Q000378:metabolism*; D064688:Metabolic Flux Analysis / Q000379:methods; D008930:Mitochondria, Liver / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects; D010168:Palmitates / Q000378:metabolism / Q000494:pharmacology*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D000975:Antioxidants; D002247:Carbon Isotopes; D010168:Palmitates; D017382:Reactive Oxygen Species; D053470:Caspases, Effector; D000111:Acetylcysteine
keywords: 2′-7′-dichlorodihydrofluorescein diacetate; BSA; CAC; Eto; FFA; GC-MS; H(2)DCFDA; Isotopomer modeling; Lipotoxicity; MFA; MUFA; Mass spectrometry; Metabolic flux analysis; N-acetyl cysteine; NAC; NAFLD; NASH; OA; PA; PHEN; PI; ROS; Reactive oxygen species; SFA; bovine serum albumin; citric acid cycle; etomoxir; free fatty acid; gas chromatography–mass spectrometry; metabolic flux analysis; monounsaturated fatty acid; non-alcoholic fatty liver disease; non-alcoholic steatohepatitis; oleate; palmitate; phenformin; propidium iodide; reactive oxygen species; saturated fatty acid.