Abstract
Inhibition by triacsins and troglitazone of long chain fatty acid incorporation into cellular lipids suggests the existence of inhibitor-sensitive and -resistant acyl-CoA synthetases (ACS, EC ) that are linked to specific metabolic pathways. In order to test this hypothesis, we cloned and purified rat ACS1, ACS4, and ACS5, the isoforms present in liver and fat cells, expressed the isoforms as ACS-Flag fusion proteins in Escherichia coli, and 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 5-Flag isoforms differed in their apparent K(m) values for ATP, thermolability, pH optima, requirement for Triton X-100, and sensitivity to N-ethylmaleimide and phenylglyoxal. The ACS inhibitor triacsin C strongly inhibited ACS1 and ACS4, but not ACS5. The thiazolidinedione (TZD) insulin-sensitizing drugs and peroxisome proliferator-activated receptor gamma (PPARgamma) ligands, troglitazone, rosiglitazone, and pioglitazone, strongly and specifically inhibited only ACS4, with an IC(50) of less than 1.5 microm. Troglitazone exhibited a mixed type inhibition of ACS4. alpha-Tocopherol, whose ring structure forms the non-TZD portion of troglitazone, did not inhibit ACS4, indicating that the thiazolidine-2,4-dione moiety is the critical component for inhibition. A non-TZD PPARgamma ligand, GW1929, which is 7-fold more potent than rosiglitazone, inhibited ACS1 and ACS4 poorly with an IC(50) of greater than 50 microm, more than 100-fold higher than was required for rosiglitazone, thereby demonstrating the specificity of TZD inhibition. Further, the PPARalpha ligands, clofibrate and GW4647, and various xenobiotic carboxylic acids known to be incorporated into complex lipids had no effect 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 catalyze the synthesis of acyl-CoAs used for triacylglycerol synthesis and that lack of inhibition of a metabolic pathway by triacsin C does not prove lack of acyl-CoA involvement. The results further suggest the possibility that the insulin-sensitizing effects of the thiazolidinedione drugs might be achieved, in part, through direct interaction with ACS4 in a PPARgamma-independent manner.
Highlights
Acyl-CoA synthetase (ACS,1 EC 6.2.1.3) catalyzes the ligation of long chain fatty acids with coenzyme A (CoA) to produce long chain acyl-CoAs [1]
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 catalyze the synthesis of acyl-CoAs used for triacylglycerol synthesis and that lack of inhibition of a metabolic pathway by triacsin C does not prove lack of acyl-CoA involvement
We focused on ACS1, ACS4, and ACS5 because these three isoforms are present in liver, a tissue that metabolizes long chain fatty acids via a variety of synthetic and degradative pathways
Summary
Inhibition by triacsins and troglitazone of long chain fatty acid incorporation into cellular lipids suggests the existence of inhibitor-sensitive and -resistant acyl-CoA synthetases (ACS, EC 6.2.1.3) that are linked to specific metabolic pathways. Inhibition of Acyl-CoA Synthetases 1, 4, and 5 adrenal, ACS4 expression increases with exposure to adrenocorticotropic hormone and to arachidonate [15] These differences in regulation, tissue distribution, and substrate preference suggest that each ACS isoform might function independently in various tissues for different metabolic purposes. The sulfo-conjugate of troglitazone appeared to be the likely inhibitory metabolite [19] These differing effects of ACS inhibitors on metabolic end products of acyl-CoA-dependent pathways suggest the presence of inhibitor-sensitive and -resistant ACSs that are linked to specific pathways. We expressed ACS1, ACS4, and ACS5 as recombinant ACS-Flag fusion proteins, and purified each ACS by Flag antibody column chromatography
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