triacsin-c and Carcinoma--Hepatocellular

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

Fatty acid-CoA ligase 4 (FACL4) is a central enzyme controlling the unesterified free arachidonic acid (AA) level in cells and the free AA is known to induce apoptosis. We have recently reported that expression of FACL4 is upregulated in about 40% of human hepatocellular carcinoma (HCC) and 50% of HCC cell lines, suggesting that FACL4 may be involved in liver carcinogenesis. In this study, we investigated whether HCC cell growth is regulated by FACL4. Immunoblot analysis showed that SNU 398 cells express very low or no detectable level of FACL4. We, therefore, transfected the SNU 398 cells with FACL4 expression vector, and clones expressing FACL4 were pooled and analyzed. We found that forced expression of FACL4 in SNU 398 promotes the growth of cells. In addition, we observed that triacsin C, a FACL4 inhibitor, inhibits the growth of Hep 3B cell line which expresses high level of endogenous FACL4. We also found that the triacsin C-mediated growth inhibition in Hep 3B cells results from the induction of apoptosis with evidence of Bcl-2 reduction. Altogether, our data show that FACL4 affects HCC cell growth and suggest that modulation of FACL4 expression/activity is an approach for treatment of HCC.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Coenzyme A Ligases; Humans; Liver Neoplasms; Proto-Oncogene Proteins c-bcl-2; Triazenes

The mechanism(s) of fatty acid uptake by liver cells is not fully understood. We applied new approaches to address long-standing controversies of fatty acid uptake and to distinguish diffusion and protein-based mechanisms. Using HepG2 cells containing an entrapped pH-sensing fluorescence dye, we showed that the addition of oleate (unbound or bound to cyclodextrin) to the external buffer caused a rapid (seconds) and dose-dependent decrease in intracellular pH (pH(in)), indicating diffusion of fatty acids across the plasma membrane. pH(in) returned to its initial value with a time course (in min) that paralleled the metabolism of radiolabeled oleate. Preincubation of cells with the inhibitors phloretin or triacsin C had no effect on the rapid pH(in) drop after the addition of oleate but greatly suppressed pH(in) recovery. Using radiolabeled oleate, we showed that its esterification was almost completely inhibited by phloretin or triacsin C, supporting the correlation between pH(in) recovery and metabolism. We then used a dual-fluorescence assay to study the interaction between HepG2 cells and cis-parinaric acid (PA), a naturally fluorescent but slowly metabolized fatty acid. The fluorescence of PA increased rapidly upon its addition to cells, indicating rapid binding to the plasma membrane; pH(in) decreased rapidly and simultaneously but did not recover within 5 min. Phloretin had no effect on the PA-mediated pH(in) drop or its slow recovery but decreased the absolute fluorescence of membrane-bound PA. Our results show that natural fatty acids rapidly bind to, and diffuse through, the plasma membrane without hindrance by metabolic inhibitors or by an inhibitor of putative membrane-bound fatty acid transporters.

Topics: beta-Cyclodextrins; Biological Transport, Active; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Membrane; Coenzyme A Ligases; Diffusion; Fatty Acids, Unsaturated; Fluoresceins; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Oleic Acid; Phloretin; Triazenes

Long chain acyl-CoA synthetase (ACSL) catalyzes the initial step in long chain fatty acid metabolism. Of the five mammalian ACSL isoforms cloned and characterized, ACSL5 is the only isoform found to be located, in part, on mitochondria and thus was hypothesized to be involved in fatty acid oxidation. To elucidate the specific roles of ACSL5 in fatty acid metabolism, we used adenoviral-mediated overexpression of ACSL5 (Ad-ACSL5) in rat hepatoma McArdle-RH7777 cells. Confocal microscopy revealed that Ad-ACSL5 colocalized to both mitochondria and endoplasmic reticulum. When compared with cells infected with Ad-GFP, Ad-ACSL5-infected cells at 24 h after infection had 2-fold higher acyl-CoA synthetase activities and 30% higher rates of fatty acid uptake when incubated with 500 microM [1-(14)C]oleic acid. Metabolism of [1-(14)C]oleic acid to cellular triacylglycerol (TAG) increased 42% in Ad-ACSL5-infected cells, but when compared with control cells, metabolism to acid-soluble metabolites, phospholipids, and medium TAG did not differ substantially. The incorporation of [1-(14)C]oleate and [1,2,3-(3)H]glycerol into TAG was similar in Ad-ACSL5-infected cells, thus indicating that Ad-ACSL5 increased TAG synthesis through both de novo and reacylation pathways. However, [1-(14)C]acetic acid incorporation into cellular lipids showed that, when compared with control cells, Ad-ACSL5-infected cells did not increase the metabolism of fatty acids that were derived from de novo synthesis. These results suggest that uptake of fatty acids into cells is regulated by metabolism and that overexpressed ACSL5 partitions exogenously derived fatty acids toward TAG synthesis and storage.

Topics: Acetic Acid; Adenoviridae; Animals; Carcinoma, Hepatocellular; Catalytic Domain; Cell Line, Tumor; Coenzyme A Ligases; Endoplasmic Reticulum; Enzyme Inhibitors; Fatty Acids; Green Fluorescent Proteins; Immunohistochemistry; Lipids; Liver; Male; Microscopy, Confocal; Mitochondrial Proteins; Oleic Acid; Phospholipids; Protein Isoforms; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Triazenes; Triglycerides

Free fatty acids and lysophosphatidylcholine (lysoPC) are the major lipids bound to human plasma albumin. The effects of fatty acids on the hepatic production of Apolipoprotein B (apo B) have been studied but those of lysoPC have not. In HepG2 cells, lysoPC increased apo B secretion in different experiments by 50-120%, but did not affect the flotation properties of secreted lipoproteins. LysoPC affected neither the cellular protein levels nor apo A-I secretion suggesting that its effect was specific to apo B. Apo B secretion was maximum after incubating cells for 6 h with 0.2 mM lysoPC as equimolar fatty acid free bovine serum albumin (BSA) complexes. LysoPC was metabolized by cells and its fatty acids were used for the synthesis of phosphatidylcholine and triglycerides (TG). Experiments were performed to understand the mechanism of lysoPC action. LysoPC increased the incorporation of 3H-glycerol into newly synthesized cellular (3-fold) and secreted (4-fold) triglycerides, and increased the synthesis (40%) and secretion (4-fold) of phospholipids. LysoPC did not affect apo B synthesis, but inhibited the intracellular degradation of apo B and increased its secretion. Triacsin C (5 microM), an inhibitor of long chain acyl-CoA synthase, completely inhibited the induction of lipid synthesis and abolished the effect of lysoPC on apo B secretion. These studies indicated that lysoPC increased apo B secretion by inducing lipid synthesis; newly synthesized lipids probably protected apo B from intracellular degradation and enhanced secretion. These studies are consistent with the hypothesis that physiologic concentrations of lysoPC can be an important modulator for hepatic apo B secretion.

Topics: Apolipoprotein A-I; Apolipoproteins B; Carcinoma, Hepatocellular; Centrifugation, Density Gradient; Fatty Acids; Glycerol; Humans; Lipids; Lysophosphatidylcholines; Phosphatidylcholines; Phospholipids; Protein Biosynthesis; Serum Albumin, Bovine; Triazenes; Triglycerides; Tumor Cells, Cultured