Palmitic acid increases apoptosis by mitochondrial pathway in hepatocytes with growth hormone deficiency

Abstract

Objective To investigate the specific molecular mechanisms of palmitic acid (PA)-induced lipid deposition in cultured hepatocytes with knockdown of growth hormone receptor (GHR). Methods Hepatic L02 cells were treated with different concentrations of PA (0, 75, 150 and 300 μmol/L) for 12, 24, and 48 h, respectively, and cell viability was measured. Then the L02 cells were transinfected with GHR-lentiviral vector (shRNAGHR group) or control vector (vector group). Oil red "O" staining and boron-dipyrromethene (BODIPY) fluorescence staining were used to observe lipid deposition. Flow cytometry was employed to detect the apoptosis and production of reactive oxygen species (ROS). JC-1 mitochondrial membrane potential assay was applied to detect the changes in membrane potential. MitoSox fluorescence was used to detect mitochondrial ROS level. Western blotting was carried out to measure mitochondrial-mediated apoptotic protein expression. After pretreated with targeted mitochondrial antioxidant, mitoTEMPO, the cells were further observed whether it could offset PA-induced changes in ROS level and apoptosis. Results PA inhibited the survival rate of the L02 cells in a concentration-dependent manner. Compared with the control group, treatment of 150 μumol/L PA for 12, 24, and 48 h resulted in significantly increased intracellular ROS level, mitochondrial ROS level and cell apoptosis in the shRNAGHR. Oil red "O" staining and BODIPY fluorescence confirmed that shRNAGHR group was more prone to lipid deposition with same dose of PA stimulation. Western blotting showed the levels of cytochrome C protein, Cleaved-Caspase 9, Cleaved-Caspase 3, and polymerase (PARP) were significantly higher in the shRNAGHR group than the vector group (P < 0.05). Pretreatment of targeted mitochondrial antioxidant mitoTEMPO significantly ameliorated shRNAGHR induced cells apoptosis. Conclusion Knockdown of GHR aggravates lipid deposition and apoptosis in hepatocytes, and PA plays an important role in the process through mitochondria/ROS pathway.

Keywords

• growth hormone receptor knockdown
• mitochondria
• reactive oxygen species
• apoptosis