triacsin-c and Fibrosarcoma

The trafficking of acyl-CoAs within cells is poorly understood. In order to determine whether newly synthesized acyl-CoAs are equally available for the synthesis of all glycerolipids and cholesterol esters, we incubated human fibroblasts with [14C]oleate, [3H]arachidonate or [3H]glycerol in the presence or absence of triacsin C, a fungal metabolite that is a competitive inhibitor of acyl-CoA synthetase. Triacsin C inhibited de novo synthesis from glycerol of triacylglycerol, diacylglycerol and cholesterol esters by more than 93%, and the synthesis of phospholipid by 83%. However, the incorporation of oleate or arachidonate into phospholipids appeared to be relatively unimpaired when triacsin was present. Diacylglycerol acyltransferase and lysophosphatidylcholine acyltransferase had similar dependences on palmitoyl-CoA in both liver and fibroblasts; thus it did not appear that acyl-CoAs, when present at low concentrations, would be preferentially used to acylate lysophospholipids. We interpret these data to mean that, when fatty acid is not limiting, triacsin blocks the acylation of glycerol 3-phosphate and diacylglycerol, but not the reacylation of lysophospholipids. Two explanations are possible: (1) different acyl-CoA synthetases exist that vary in their sensitivity to triacsin; (2) an independent mechanism channels acyl-CoA towards phospholipid synthesis when little acyl-CoA is available. In either case, the acyl-CoAs available to acylate cholesterol, glycerol 3-phosphate, lysophosphatidic acid and diacylglycerol and those acyl-CoAs that are used by lysophospholipid acyltransferases and by ceramide N-acyltransferase must reside in two non-mixing acyl-CoA pools or, when acyl-CoAs are limiting, they must be selectively channelled towards specific acyltransferase reactions.

Topics: Acyl Coenzyme A; Acylation; Animals; Arachidonic Acid; Cholesterol Esters; Coenzyme A Ligases; Depression, Chemical; Diglycerides; Fatty Acids; Fibroblasts; Fibrosarcoma; Glycerides; Glycerol; Humans; Liver; Lysophospholipids; Mice; Phospholipids; Skin; Triazenes; Triglycerides; Tumor Cells, Cultured

Triacsins A, B, C, and D are newly discovered compounds isolated from the culture filtrate of streptomyces which are known to inhibit nonspecific long chain acyl-CoA synthetase (EC 6.2.1.3.). These inhibitors have not been previously studied with regard to their effects on arachidonoyl-CoA synthetase, an enzyme which specifically utilizes arachidonate and other icosanoid precursor fatty acids. To explore this question, we used triacsin C, a potent inhibitor of the nonspecific acyl-CoA synthetase. Triacsin C was found to inhibit the action of arachidonoyl-CoA synthetase and the nonspecific enzyme in sonicates of HSDM1C1 mouse fibrosarcoma cells. Importantly, however, the triacsin concentration and length of pre-incubation with the enzymes could be adjusted to almost completely inhibit (greater than 80%) the nonspecific long chain acyl CoA-synthetase, with less than 20% inhibition of arachidonoyl-CoA synthetase. Using intact cultured cells exposed to 1 ug/ml triacsin for up to 15 minutes, we unexpectedly observed preferential inhibition of arachidonoyl-CoA synthetase activity. In intact cell studies, arachidonoyl-CoA synthetase was inhibited greater than 90%, with 55-60% inhibition of the nonspecific acyl-CoA synthetase. As additional evidence of its inhibition of acyl-CoA synthetase enzymes in intact cells, triacsin C inhibited both fatty acid uptake into cells and icosanoid production, metabolic processes which in certain cell types appear to be dependent on acyl-CoA synthetase activity. Thus, triacsin C is a novel inhibitor which can alter the fatty metabolism of intact cells. This compound can be of significant value in determining the specific cellular functions of the two acyl-CoA synthetase enzymes.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Biological Transport; Cell Line; Cell Membrane; Coenzyme A Ligases; Dinoprostone; Fibrosarcoma; Mice; Oleic Acid; Oleic Acids; Repressor Proteins; Saccharomyces cerevisiae Proteins; Triazenes