Expression and characterization of recombinant rat acyl-CoA synthetases 1,4, and 5 - Selective inhibition by triacsin C and thiazolidinediones

Inhibition by triacsins and troglitazone of long chain fatty acid incorpora tion into cellular lipids suggests the existence of inhibitor-sensitive and -resistant acyl-CoA synthetases (ACS, EC 6.2.1.3) that are linked to speci fic metabolic pathways. In order to test this hypothesis, we cloned and pur ified rat ACS1, ACS4, and ACS5, the isoforms present in liver and fat cells , expressed the isoforms as ACS-Flag fusion proteins in Escherichia coli, a nd purified them by Flag affinity chromatography, The Flag epitope at the C terminus did not alter the kinetic properties of the enzyme. Purified ACS1 -, 4-, and 8-Flag isoforms differed in their apparent K-m values for ATP, t hermolability, pH optima, requirement for Triton X-100, and sensitivity to N-ethylmaleimide and phenylglyoxal, The ACS inhibitor triacsin C strongly i nhibited ACS1 and ACS4, but not ACS5, The thiazolidinedione (TZD) insulin-s ensitizing drugs and peroxisome proliferator-activated receptor gamma (PPAR gamma) ligands, troglitazone, rosiglitazone, and pioglitazone, strongly an d specifically inhibited only ACS(4), with an IC50 of less than 1.5 muM. Tr oglitazone exhibited a mixed type inhibition of ACS4. alpha -Tocopherol, wh ose ring structure forms the non-TZD portion of troglitazone, did not inhib it ACS4, indicating that the thiazolidine-2,4-dione moiety is the critical component for inhibition. A non-TZD PPAR gamma ligand, GW1929, which is 7-f old more potent than rosiglitazone, inhibited ACS1 and ACS4 poorly with an IC50 of greater than 50 muM, more than 100-fold higher than was required fo r rosiglitazone, thereby demonstrating the specificity of TZD inhibition. F urther, the PPAR alpha ligands, clofibrate and GW4647, and various xenobiot ic carboxylic acids known to be incorporated into complex lipids had no eff ect on ACS1, -4, or -5, These results, together with previous data showing that triacsin C and troglitazone strongly inhibit triacylglycerol synthesis compared with other metabolic pathways, suggest that ACS1 and ACS4 catalyz e the synthesis of acyl-Coks used for triacylglycerol synthesis and that la ck of inhibition of a metabolic pathway by triacsin C does not prove lack o f acyl-CoA involvement. The results further suggest the possibility that th e insulin-sensitizing effects of the thiazolidinedione drugs might be achie ved, in part, through direct interaction with ACS4 in a PPAR gamma -indepen dent manner.