(182b) A Systems Approach to Identify Metabolic Pathways Controlling Hepatic Lipotoxicity

In the past half century, the prevalence of diabetes and obesity has grown significantly, a trend which has been described as a health epidemic in the US. The similar pathogenesis of these two diseases involves abnormal accumulation of lipids (steatosis) in non-adipose tissues due to elevated free fatty acids. In the liver, saturated fatty acids (SFA) overexposure has been shown to induce ER stress and oxidative stress in hepatocytes, leading to increased apoptosis. However, the exact identity of the lipid species responsible for these effects is still largely uncertain, and the mechanism by which ER and redox homeostasis becomes dysregulated remains unknown. 

In order to better characterize the phenotypic response of hepatic cells to SFA overexposure, we have sought to systematically perturb the main routes of fatty acid utilization and disposal, including β-oxidation, triglyceride (TG) synthesis and phosphotidylcholine (PC) synthesis. Experimentally, H4IIEC3 hepatic cells are incubated with varying concentrations of SFA (palmitate, 200-400 µM) in combination with treatments designed to prevent reactive oxygen species (ROS) accumulation and lipoapoptosis by modulating specific lipid metabolic pathways. Etomoxir and AICAR have been shown previously to inhibit and up-regulate β-oxidation, respectively. While etomoxir did not rescue ROS or viability in palmitate-treated cells, AICAR limited ROS production and prevented cell death at concentrations between 250-500 µM. Phospholipase inhibitors have also been applied to modulate PC turnover. Phospholipase A₂ inhibitor PACOCF₃ demonstrated a dose-dependent reduction of ROS and caspase 3/7 activity. Preliminary results indicate that phospholipase C inhibitor U73122 can also successfully reduce ROS accumulation and rescue the 24-hour viability of H4IIEC3 cells.

Our ongoing work is focused on a detailed investigation of the mechanisms by which AICAR, PACOCF3 and U73122 are able to reverse the lipotoxic effects of SFA overexposure. We are applying lipidomic profiling and metabolic flux analysis (MFA) based on isotopic enrichment with ¹³C-labeled palmitate to understand the metabolic fate of exogenous SFA and how it is altered by these interventions. This involves HPLC separation and GC-MS analysis of lipid extracts, followed by computational analysis of lipidomic and isotopic labeling data to identify pathways that are responsible for the observed metabolic phenotypes. Developing a better understanding of the mechanisms of SFA overexposure leading to ROS accumulation and lipoapotosis, and the identity of the specific lipid species mediating these events, will lead to more efficient and targeted therapies for treatment of fatty liver disease and related complications associated with diabetes and obesity.