Mitochondrial response to lipotoxic and mitotoxic stress in human hepatocellular carcinoma cell line (HEPG2)

As dietary saturated fatty acid intake increases, the prevalence of obesity, type 2 diabetes (T2DM) and non-alcoholic fatty liver disease (NAFLD) increases. The common point in the pathogenesis of these diseases is that the increased amount of free fatty acids begins to accumulate in non-adipose tissues such as liver, skeletal muscle, heart and pancreas. This condition, defined as lipotoxicity, causes organelle dysfunction, cell damage, metabolic disorders, chronic inflammation, loss of cellular function and death of non-adipose tissue with increasing lipid accumulation. When stress occurs in the mitochondria due to lipotoxicity, lipid peroxidation, ROS increase, and consequently cell death are triggered. Palmitic Acid (PA) is the most saturated free fatty acid found in diet and serum. It is a long chain fatty acid with 16 carbons. Although the underlying mechanism is not fully known, the conversion of fatty acids to triglycerides (TG) causes cytotoxicity. Since the conversion of PA to TG is less, it shows more toxic properties. Another source of stress is carbonyl cyanide 3-chlorophenylhydrazone (CCCP). CCCP is an agent that causes mitotoxicity in cells and is widely used in mitophagy studies. In the light of all these data, the aim of this study was to investigate the difference in the stress response of mitochondria by applying lipotoxic and protonophore stress separately in human HepG2 cell line. Thus, it is aimed to obtain more detailed information about the proteins involved in the responses of mitochondria to autophagy, mitophagy against different stimuli. In addition, the effect of duration and variety of stress applied on response difference was investigated. For this purpose, expression of CHOP, HSP10, HSP60, Parkin, which are mitochondria-related stress and mithophagy genes, and Optineurin, HACE1, TBK1, p62 and LC3-II genes that act as adapters/ receptors in autophagy were examined. As a result, cellular stress response started and decreased earlier in the PA group; In the CCCP group, it was observed that it started later and lasted longer. In the PA group, the most effective time was 12 hours, whereas in the CCCP group it was 24 hours, especially the expression of CHOP gene in mitochondrial stress and Parkin, OPTN, HACE1, TBK1, P62 and LC3-II genes in mitochondrial stress were increased.