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. Author manuscript; available in PMC: 2012 Mar 1.
Published in final edited form as: Biochem Pharmacol. 2010 Dec 22;81(5):649–660. doi: 10.1016/j.bcp.2010.12.014

Figure 10. Dose effect of soraphen A on microsomal fatty acid elongase activity and HepG2 lipid synthesis, fatty acid elongation & PUFA synthesis.

Figure 10

Panel A: Dose effect of soraphen A on fatty acid elongase activity in isolated hepatic microsomes. Mouse liver microsomes were isolated and used for a fatty acid elongase assay (Methods) [24, 25]. Microsomes were pretreated with DMSO or soraphen A (at 0.1 to 10 µM) for 10 mins prior to initiating the reaction with the addition of NADPH. After a 20 minute reaction, the elongation products were isolated and quantified [24, 25]. Results are expressed as % Control, mean ± SD, n=4. Elongase activity in control microsomes was 6 ± 0.8 nmoles malonyl CoA assimilated into fatty acid/mg protein, a value comparable to previously reported values [24, 25].

Panel B. Dose response of soraphen A regulation of lipid synthesis & fatty acid elongation. HepG2 cells were pre-treated with soraphen A, ranging from 0.1 nM to 1 µM, 2 hour prior to adding [2-14C]-acetate (lipid synthesis) or [1-14C]-18:2,n-6 [50 µM) (fatty acid elongation) as described above for all labeling studies. Cells were harvested and extracted for total lipid 6 hrs after adding [2-14C]-acetate or [1-14C]-18:2,n-6. The evaluation of soraphen A effects on lipid synthesis and fatty acid elongation used cells that were not infected with recombinant adenovirus. Lipid synthesis was assessed by quantifying [2-14C]-acetate assimilation into organic extracts; this includes both fatty acids and cholesterol. Fatty acid elongation was quantified by measuring the amount of 14C-20:2,n-6 formed following [1-14C]-18:2,n-6 treatment. Results are expressed as % DMSO control, mean ± SD, n=3.

Panel C & D: Dose response of soraphen A on PUFA synthesis. To evaluate soraphen A effects on PUFA synthesis, HepG2 cells were infected with Ad-FADS1 and Ad-FADS2 48 hrs prior to adding soraphen A and [1-14C]-18:2,n-6, as described in Fig. 6. PUFA synthesis was quantified by measuring the amount of [1-14C]-18:2,n-6 converted to 14C20–22 PUFA (Panel C) and 14C-18:3,n-6 (Panel D). 14C20–22 PUFA is the sum of 14C assimilated into 20:2,n-6, 20:3,n-6, 20:4,n-6, 22:4,n-6, 22:5,n-6. Results are expressed as % DMSO-treated cells. Results are from 2 separate studies with duplicate samples, mean ± S.D. n=4.