triacsin-c and etomoxir

Free fatty acids (FFAs) are proposed to play a pathogenic role in both peripheral and hepatic insulin resistance. We have examined the effect of saturated FFA on insulin signalling (100 nM) in two hepatocyte cell lines. Fao hepatoma cells were treated with physiological concentrations of sodium palmitate (0.25 mM) (16:0) for 0.25-48 h. Palmitate decreased insulin receptor (IR) protein and mRNA expression in a dose- and time-dependent manner (35% decrease at 12 h). Palmitate also reduced insulin-stimulated IR and IRS-2 tyrosine phosphorylation, IRS-2-associated PI 3-kinase activity, and phosphorylation of Akt, p70 S6 kinase, GSK-3 and FOXO1A. Palmitate also inhibited insulin action in hepatocytes derived from wild-type IR (+/+) mice, but was ineffective in IR-deficient (-/-) cells. The effects of palmitate were reversed by triacsin C, an inhibitor of fatty acyl CoA synthases, indicating that palmitoyl CoA ester formation is critical. Neither the non-metabolized bromopalmitate alone nor the medium chain fatty acid octanoate (8:0) produced similar effects. However, the CPT-1 inhibitor (+/-)-etomoxir and bromopalmitate (in molar excess) reversed the effects of palmitate. Thus, the inhibition of insulin signalling by palmitate in hepatoma cells is dependent upon oxidation of fatty acyl-CoA species and requires intact insulin receptor expression.

Topics: Aminoimidazole Carboxamide; Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Enzyme Activation; Enzyme Inhibitors; Epoxy Compounds; Extracellular Signal-Regulated MAP Kinases; Fatty Acids; Fatty Acids, Nonesterified; Forkhead Transcription Factors; Glycogen Synthase Kinase 3; Hypoglycemic Agents; Insulin; Insulin Receptor Substrate Proteins; Liver; Liver Neoplasms; Mice; Mice, Knockout; Oxidation-Reduction; p38 Mitogen-Activated Protein Kinases; Palmitic Acid; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Receptor, Insulin; Ribonucleotides; Signal Transduction; Triazenes

Generation of reactive oxygen species may contribute to the pathogenesis of diseases involving intracellular lipid accumulation. To explore the mechanisms leading to these pathologies we tested the effects of etomoxir, an inhibitor of carnitine palmitoyltransferase I which contains a fatty acid-derived structure, in C2C12 skeletal muscle cells. Etomoxir treatment for 24 h resulted in a down-regulation of peroxisome proliferator-activated receptor alpha (PPARalpha) mRNA expression, achieving an 87% reduction at 80 microm etomoxir. The mRNA levels of most of the PPARalpha target genes studied were reduced at 100 microm etomoxir. By using several inhibitors of de novo ceramide synthesis and C(2)-ceramide we showed that they were not involved in the effects of etomoxir. Interestingly, the addition of triacsin C, a potent inhibitor of acyl-CoA synthetase, to etomoxir-treated C2C12 skeletal muscle cells did not prevent the down-regulation in PPARalpha mRNA levels, suggesting that the active form of the drug, etomoxir-CoA, was not involved. Given that saturated fatty acids may generate reactive oxygen species (ROS), we determined whether the addition of etomoxir resulted in ROS generation. Etomoxir increased ROS production and the activity of the well known redox transcription factor NF-kappaB. In the presence of the pyrrolidine dithiocarbamate, a potent antioxidant and inhibitor of NF-kappaB activity, etomoxir did not down-regulate PPARalpha mRNA in C2C12 skeletal muscle cells. These results indicate that ROS generation and NF-kappaB activation are responsible for the down-regulation of PPARalpha and may provide a new mechanism by which intracellular lipid accumulation occurs in skeletal muscle cells.

Topics: Animals; Cell Line; Cells, Cultured; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Inhibitors; Epoxy Compounds; Fatty Acids; Flavonoids; Flow Cytometry; Mice; Muscle, Skeletal; NF-kappa B; Polymerase Chain Reaction; Protein Binding; Reactive Oxygen Species; Receptors, Cytoplasmic and Nuclear; RNA; RNA, Messenger; Sphingosine; Thiocarbamates; Time Factors; Transcription Factors; Triazenes