The overall objective of the current application is to elucidate the mechanism by which metabolic changes activate apoptotic signaling in hepatic cells exposed to elevated saturated fatty acids (SFAs).The central hypothesis is that endoplasmic reticulum (ER) stress induced by SFAs leads to altered coupling between central carbon pathways, which subsequently triggers mitochondrial accumulation of reactive oxygen species (ROS) resulting in apoptosis. The rationale for the proposed research is that, once it is known how metabolic pathway fluxes impact lipid-induced apoptosis, these pathways can be modulated by therapeutic interventions that target the underlying metabolic factors causing lipotoxicity. The plan is to test the central hypothesis by pursuing the following specific aims. First, to determine the mechanism by which SFAs induce ROS accumulation in hepatic cells. Second, to identify the lipid intermediate responsible for inducing ER stress in SFA-treated hepatic cells. These aims will be accomplished using a systems approach that focuses on integrated metabolic pathways versus individual reactions and where the concept of the metabolic network is of central importance. The approach involves both GC-MS metabolic profiling to track the relative concentrations and isotopic labeling patterns of many different intracellular metabolites simultaneously, as well as metabolic flux analysis (MFA) to reconstruct comprehensive flux maps that represent the dynamic flow of material throughout the network. By subjecting hepatic cells to a variety of treatments aimed at perturbing lipid and central carbon metabolism, they will dissect the pathways involved in SFA-induced lipotoxicity.
