Palmitate-induced Activation of Mitochondrial Metabolism Promotes Oxidative Stress and Apoptosis in H4IIEC3 Rat Hepatocytes |
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Authors: | Robert A. Egnatchik Alexandra K. Leamy Yasushi Noguchi Masakazu Shiota Jamey D. Young |
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Affiliation: | 1. Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN, USA;2. Institute for Innovation, Ajinomoto Co., Inc., Kawasaki, Japan;3. Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, USA |
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Abstract: | ObjectiveHepatic 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.Materials/MethodsWe 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 13C metabolic flux analysis (MFA) to quantify mitochondrial pathway alterations associated with these treatments.ResultsTreatment 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, 13C 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.ConclusionOur 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. |
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Keywords: | BSA, bovine serum albumin CAC, citric acid cycle Eto, etomoxir FFA, free fatty acid GC-MS, gas chromatography&ndash mass spectrometry H2DCFDA, 2&prime -7&prime -dichlorodihydrofluorescein diacetate MFA, metabolic flux analysis MUFA, monounsaturated fatty acid NAC, N-acetyl cysteine NAFLD, non-alcoholic fatty liver disease NASH, non-alcoholic steatohepatitis OA, oleate PHEN, phenformin PA, palmitate PI, propidium iodide ROS, reactive oxygen species SFA, saturated fatty acid. |
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