The relationship between oxidative stress, triglyceride accumulation and mitochondrial function in in vitro model of hepatocellular steatosis

• Main Author: Lockman, Khalida Ann
• Published: University of Edinburgh 2013
• Subjects: 616.3
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.726484

There is still debate about the relationship between fat accumulation and mitochondrial function in nonalcoholic fatty liver disease. It is a critical question since a proportion of individuals with steatosis progress to steatohepatitis. This thesis is focused on defining i) the effect of triglyceride accumulation and reactive oxygen species (ROS) on mitochondrial function ii) the contribution of triglyceride, ROS and subsequent mitochondrial impairment to the metabolism of energy substrates iii) the effect of the intracellular antioxidant, N-acetylcysteine, on hepatic mitochondrial function and metabolic alterations associated with human steatohepatitis iv) the effect of different free fatty acids species on ROS production and subsequent metabolic response in hepatocytes. To address these questions, we designed in vitro models using human hepatoblastoma C3A cells treated with various combinations of oleate, octanoate (O), lactate (L), pyruvate (P) and ammonia (N) acutely or for 72 hours, before measurements of triglyceride concentration, cell respiration, ROS production, mitochondrial membrane potential, ketogenesis and gluconeogenesis, metabolomics analyses, confocal and electron microscopy. Acutely, LPON treatment enhanced mitochondrial respiration and ROS formation. After 72 hours, despite the similarities in triglyceride accumulation, LPON treatment, but not oleate, dramatically affected mitochondrial function as evidenced by decreased respiration, increased mitochondrial membrane potential and enhanced ketogenesis. Importantly, this was associated with increased markers of oxidative stress and enhanced gluconeogenesis. Furthermore, reduction in triglyceride with metformin did not improve mitochondrial function. By comparison, respiration and ROS formation remained unperturbed with oleate. The addition of the antioxidant N-acetylcysteine prevented mitochondrial dysfunction and reversed metabolic changes seen with LPON, strongly suggesting ROS involvement in mediating mitochondrial impairment. Our data indicate that increased ROS formation, rather than cellular steatosis per se, impairs mitochondrial function. Thus, reduction in cellular steatosis may not always be the desired outcome without concomitant improvement in mitochondrial function and/or a decrease in ROS formation.