Abstract
To identify metabolic pathways involved in hepatic lipoapoptosis, metabolic flux analysis using [U-(13)C(5)]glutamine as an isotopic tracer was applied 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 citric acid cycle (CAC) flux and glutamine uptake. This decoupling of glycolysis and CAC fluxes occurred during the period following palmitate exposure but preceding the onset of apoptosis. Oleate co-treatment restored most fluxes to their control levels, resulting in steatotic lipid accumulation while preventing apoptosis. In addition, palmitate strongly increased the cytosolic NAD(+)/NADH ratio, whereas oleate co-treatment had the opposite effect on cellular redox. We next examined the influence of amino acids on these free fatty acid-induced phenotypic changes. Increased medium amino acids enhanced reactive oxygen species (ROS) generation and apoptosis in PA-cells but not 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 indicate that free fatty acid-induced ROS generation and apoptosis are accompanied by the decoupling of glycolysis and CAC fluxes leading to abnormal cytosolic redox states. Amino acids play a modulatory role in these processes via a mechanism that does not involve ceramide accumulation.
Highlights
Titis and correlates with disease severity [1]
A consensus mechanism linking saturated fatty acids (SFAs)-induced metabolic alterations to apoptosis has been difficult to establish. To further address these questions, we hypothesized that metabolites or pathways other than those involved with ceramide synthesis may influence reactive oxygen species (ROS) generation and the resulting cellular phenotypes associated with elevated SFAs
We hypothesized that altering amino acid levels could be one way to modulate the response of liver cells to elevated free fatty acids (FFAs) concentrations, thereby providing additional insight into the metabolic deviations leading to apoptosis
Summary
Materials—Fumonisin-B1, cycloserine, sphingomyelinase (Bacillus cereus) and free amino acids were purchased from Sigma. Cells were preconditioned for 6 h in unlabeled medium before washing with phosphate-buffered saline and replenishing with medium containing labeled glutamine Both medium and cellular metabolites were analyzed by GC-MS at 3, 6, and 9 h. Non-polar Metabolite Analysis—For ceramide and free fatty acid analysis, dried non-polar samples were dissolved in 1.55 ml of isooctane/methanol/ethyl acetate (20:10:1). For total cellular fatty acid analysis, dried non-polar samples were dissolved in 250 l of 0.5 N KOH in methanol and incubated at 70 °C for 1 h. Metabolic Flux Determination—The labeling patterns of metabolic intermediates and by-products that emerge upon feeding an isotopically labeled substrate to cultured cells are directly determined by the relative pathway fluxes in their biochemical network [28] This enables the reconstruction of metabolic flux maps by minimizing the lack of fit between simulated and measured GC-MS data. Statistical significance was determined by Dunnett’s or Tukey’s honestly significant differences test according to each experimental design
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