Cell dysfunction and death induced by elevated serum free fatty acid (FFA) concentrations is a key contributor to the pathogenesis of obesity and type 2 diabetes. In particular, fat accumulation in the liver can lead to a condition known as non-alcoholic steatohepatitis (NASH), which involves tissue inflammation and damage that may culminate in cirrhosis. Hepatic apoptosis is a prominent feature of NASH and correlates with disease severity. Previous in vitro studies have demonstrated that saturated fatty acids (SFAs; e.g., palmitate) induce reactive oxygen species (ROS) generation and apoptosis, while mono-unsaturated fatty acids (e.g., oleate) lead to lipid accumulation but do not trigger apoptosis. Ceramide accumulation has been considered a primary factor responsible for these effects, because ceramide is synthesized de novo from palmitate and serine and also has been shown to activate apoptotic signaling. Recent studies, however, have reported that SFAs can induce apoptosis through ROS formation and endoplasmic reticulum stress without altering intracellular ceramide levels. Thus, a consensus mechanism linking SFA-induced metabolic alterations to apoptosis has been difficult to establish.

To identify metabolic pathways causing hepatic lipoapoptosis, we applied metabolic flux analysis (MFA) using [U-¹³C₅]-glutamine as an isotopic tracer to quantify phenotypic changes in H4IIEC3 hepatoma cells treated with either palmitate alone (PA-cells) or both palmitate and oleate in combination (PA/OA-cells). Our results indicate that palmitate inhibited glycolysis and lactate dehydrogenase fluxes while activating TCA cycle flux and glutamine uptake. This decoupling of glycolysis and TCA cycle fluxes occured during the period following palmitate exposure but preceding the onset of apoptosis. Oleate co-treatment restored most fluxes to their control levels, resulting in increased lipid accumulation while preventing apoptosis. In addition, palmitate strongly increased the cytosolic NAD⁺/NADH ratio, while oleate co-treatment had the opposite effect on cellular redox.

We next examined the influence of amino acids on these FFA-induced phenotypic changes. Increased medium amino acids enhanced ROS generation and apoptosis in PA-cells, but these effects were not observed in PA/OA-cells. Overloading the medium with non-essential amino acids induced apoptosis, but essential amino acid overloading partially ameliorated apoptosis. Glutamate was the most effective single amino acid in promoting ROS. Amino acid overloading also increased cellular palmitoyl-ceramide; however, ceramide synthesis inhibitors had no effect on measurable indicators of apoptosis. Our results therefore suggest that FFA-induced ROS generation and apoptosis are accompanied by the decoupling of glycolysis and TCA cycle fluxes leading to abnormal cytosolic redox states. Amino acids play a modulatory role in these processes, not through ceramide, but possibly through the control of TCA cycle flux.