title: ER calcium release promotes mitochondrial dysfunction and hepatic cell lipotoxicity in response to palmitate overload.
abstract: Palmitate overload induces hepatic cell dysfunction characterized by enhanced apoptosis and altered citric acid cycle (CAC) metabolism; however, the mechanism of how this occurs is incompletely understood. We hypothesize that elevated doses of palmitate disrupt intracellular calcium homeostasis resulting in a net flux of calcium from the ER to mitochondria, activating aberrant oxidative metabolism. We treated primary hepatocytes and H4IIEC3 cells with palmitate and calcium chelators to identify the roles of intracellular calcium flux in lipotoxicity. We then applied (13)C metabolic flux analysis (MFA) to determine the impact of calcium in promoting palmitate-stimulated mitochondrial alterations. Co-treatment with the calcium-specific chelator BAPTA resulted in a suppression of markers for apoptosis and oxygen consumption. Additionally, (13)C MFA revealed that BAPTA co-treated cells had reduced CAC fluxes compared to cells treated with palmitate alone. Our results demonstrate that palmitate-induced lipoapoptosis is dependent on calcium-stimulated mitochondrial activation, which induces oxidative stress.
authors: Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN, USA.
authors: Robert A
authors: RA
authors: Egnatchik
authors: Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN, USA.
authors: Alexandra K
authors: AK
authors: Leamy
authors: Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, USA.
authors: David A
authors: DA
authors: Jacobson
authors: Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, USA.
authors: Masakazu
authors: M
authors: Shiota
authors: Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN, USA ; Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, USA.
authors: Jamey D
authors: JD
authors: Young
keywords: APE, atom percent enrichment; BSA, bovine serum albumin; CAC, citric acid cycle; ER stress; FFA, free fatty acid; Fatty liver; GC–MS, gas chromatography–mass spectrometry; H2DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate; Lipotoxicity; MFA, metabolic flux analysis; MUFA, monounsaturated fatty acid; Metabolic flux analysis; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; OA, oleate; Oxidative stress; PA, palmitate; PI, propidium iodide; ROS, reactive oxygen species; SERCA, sarcoplasmic-endoplasmic reticulum calcium ATPase; SFA, saturated fatty acid