A multiplexed cell assay in Hepg2 cells for the identification of delta-5, delta-6, and delta-9 desaturase and elongase inhibitors

Abstract

A multiplexed cell assay has been optimized, to measure the activities of fatty acyl-CoA elongase, delta-5 desaturase (Δ5D), delta-6 desaturase (Δ6D), and delta-9 desatorase (Δ9D) together using 14C-labeled tracers in HepG2 cells, which express the human stearoyl-CoA desaturase-1 isoform (SCDl) exclusively. The Δ5 and Δ9 desaturase activities are indexed by the efficient conversion of [l- 14C]-eicosatrienoic acid (C20:3, cis-8,11,14) to 14C- arachidonic acid (C20:4, cis-5,8,11,14) and the conversion of [l- 14C]-stearic acid to 14C-oleic acid (C 18:1, cis-9), respectively. CP-74006 potently blocks the Δ5D activity with an IC 50 value of 20 nM and simplifies the metabolism of [l- 14C]-a-linolenate (C18:3, cis-9,12,15) by accumulating 14C-eicosatetraenoic acid (C20:4, cis-8,11,14,17) as the major 14C-eicosatrienoic acid (C20:3, cis-11,14,17) and 14C-docosatetraenoic acid (C22:4, cis-10,13,16,19) as the minor metabolites through Δ6 desaturation and elongation. This simplified, metabolite spectrum enables the delineation of the Δ6D activity by comparing the combined Δ6D/elongase activity index of the 14C-(C20:4/C18:3) ratio with the corresponding elongation index of the 14C-(C20:3/C18:3) ratio following compound treatment. SC-26196 and sterculic acid specifically inhibit the Δ6D and Δ9D activities with an IC50 value of 0.1 μM and 0.9 μM, respectively. This medium-throughput cell assay provides an efficient tool in the identification of specific desaturase and elongase inhibitors. © 2010 Society for Biomolecular Sciences.