HepG2 and LnCap were grown to 80% confluence as described in the Methods and treated with DMSO or soraphen A (100 nM) for 2 hrs prior to addition of ¹⁴C-16:0 (50 µM). Cells were maintained in the presence of vehicle (DMSO) or soraphen A (100 nM) for an additional 6 hrs. After treatment, cells were extracted for total lipid, saponified and fractionated by RP-HPLC as described in Methods. Panels A & C illustrate the distribution of ¹⁴C in 16:0 and its elongation and desaturation products in HepG2 cells [A] and LnCap cells [C]. Results are reported as % Distribution of ¹⁴C in Fatty Acids, mean ± SD, n=4; *, p≤0.05 DMSO (white bars) versus soraphen A (black bars). Panels B and D represent the desaturation and elongation index calculated from the data in Panels A and C, respectively. The desaturation index was calculated by summing the ¹⁴C-CPS in [16:1,n-7 + 18:1,n-7 + 18:1,n-9] and dividing by the sum of ¹⁴C-CPS in [16:0 + 18:0]. The elongation index was calculated by summing the ¹⁴C-CPS in [18:0 + 18:1,n-7 + 18:1,n-9] and dividing by the sum of ¹⁴C-CPS in [16:0 + 16:1,n-7].

A. Pathway for linoleate metabolism [42, 62]. B. HepG2 cells were treated with soraphen A (100 nM) for 2 hrs before adding ¹⁴C-18:2,n-6 (50 µM). Six hours later cells were harvested for total lipid extraction, saponification, and RP-HPLC analysis. Results are reported at % Distribution of ¹⁴C in 18:2,n-6 products; mean ± SD, n=3, t-test.

Panel A: Infection of HepG2 with recombinant adenovirus expressing FADS1 (Ad-FADS1) and FADS2 (Ad-FADS2). HepG2 cells were infected with Ad-FADS1 and Ad-FADS2 at 20 PFU/cell each. The effect of Ad-FADS1, Ad-FADS2 and the combination of Ad-FADS1 & Ad-FADS2 on 18:2,n-6 metabolism in HepG2 cells is illustrated in Fig. 4S, Supplementary data. Forty-eight hours after adenoviral infection, HepG2 cells were treated with DMSO (white bars), soraphen A (black bars) and [1-¹⁴C]-18:2,n-6 (50 µM) as described in Fig. 5. Six hours later, cells were harvested for lipid extraction, saponification and fractionation. Results are expressed as % Distribution of ¹⁴C Fatty Acids; mean ± SD, n=3; *, p≤0.05 DMSO versus soraphen A. Panel B: Effect of soraphen A on 18:2,n-6 metabolism in LnCap cells. LnCap cells were treated with DMSO (white bars) or soraphen A (black bars) for 2 hrs prior to the addition of ¹⁴C-18:2,n-6 (50 µM). Six hours afterward, cells were harvested for extraction, saponification and RP-HPLC fractionation. Results are expressed as % Distribution of ¹⁴C Fatty Acids; mean ± SD, n=3, *, p≤0.05 DMSO versus soraphen A, t-test.

HepG2 cells were infected with Ad-Luc or Ad-Elovl5 at 10–200 PFU/cell 48 hrs before fatty acid treatment. [Panel A]: Levels of Elovl5 were quantified by immunoblot (Methods). [Panel B]: Cells were treated with 50 µM [1-¹⁴C]-linoleate for 6 hrs and harvested for total lipid extraction and saponification for RP-HPLC fractionation and quantitation of ¹⁴C-20:2,n-6. Results are presented as 20:2,n-6, % of Total ¹⁴C-fatty acid recovered by RP-HPLC. [Panel C]: Media was harvested for analysis of ¹⁴C-linoleic acid oxidation products, i.e., acid soluble material (ASM, nmoles/mg protein). Results are the mean ± range of 2 separate studies. *. P≤0.05 versus DMSO, t-test.

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.

A: Measurement of malonyl CoA in HepG2 cells. HepG2 cells were grown to ~90% confluence in 100 mm Petri dishes in DMEM + 10% FBS. Cells were treated with DMSO (0.5%, vehicle for soraphen A] or soraphen A (100 nM) for 6 hrs, harvested and extracted for malonyl CoA (Methods). Results are expressed as Malonyl CoA, pmoles/mg protein; the results are the mean + standard deviation (S.D.); n = 5; *; p<0.01, t-test.
B & C: Effect of soraphen A on de novo lipogenesis in HepG2 cells. HepG2 cells grown in 6 well plates were treated with DMSO (white bars) or soraphen A (100 nM, black bars) for 2 hrs. prior to adding [2-¹⁴C]-acetate (0.5 µCi/well-6 well plate; 4.3 µM; 58 mCi/mmole). Cells were maintained in media containing DMSO or soraphen A for an additional 6 hrs and then extracted for total lipids (panel B) or fatty acids (panel C) (Methods). Total lipid was fractionated by thin layer chromatography & the distribution of ¹⁴C in lipid fractions was quantified by phosphor image analysis. Lipid standards (cholesterol ester [CE, 18:1,n-9], triacylglycerol [TAG, triolean], NEFA [18:1,n-9], cholesterol (Chol), diacylglycerol [DAG, diolean] and polar lipid [PL, phosphatidyl choline] were obtained from Nu-Chek Prep & Avanti Polar Lipids. Results are expressed as phosphor-image units × 10⁻⁴ mean ± SD, n=3; *, p≤0.05 DMSO (white bars) versus soraphen A (black bars). Total lipids were saponified and the resulting non-esterified fatty acids were fractionated and quantified by RP-HPLC and β-scintillation counting. Results are expressed as Distribution of ¹⁴C-in fatty acids, CPS (counts/second) mean ± SD, n=3; *, p≤0.05 DMSO (white bars) versus soraphen A (black bars).

HepG2 cells were infected with Ad-Luc (control virus) or Ad-Elovl6 at 20 PFU/cells 48 hrs prior to treatment with [1-¹⁴C]-16:0 (Methods). The immunoblot (Panel A) represents the level of Elovl6 protein abundance in HepG2 following infection with Ad-Luc or Ad-Elovl6. Ad-Luc has no effect on Elovl6 protein abundance; Ad-Elovl6 infection increases Elovl6 ~3-fold, a value consistent with physiological changes seen in vivo [24, 25]. Panel B: Soraphen effects on palmitate metabolism in HepG2 cells with elevated Elovl6 expression. Forty-eight hours after adenoviral infection, cells were treated with [1-¹⁴C]-16:0 (50 µM) and DMSO or Soraphen A as described above. Six hours after treatment cells were harvested for extraction and saponification for RP-HPLC fractionation. Treatments: Ad-Luc + DMSO (white bars); Ad-Luc + soraphen A (light gray bars); Ad-Elovl6 + DMSO (black bars); Ad-Elovl6 + soraphen A (dark gray bars). Results in panels A, B and D are expressed as % Distribution of ¹⁴C Fatty Acids; mean ± SD, n=3; *, p≤0.05 DMSO versus soraphen A; #, p≤ 0.05 Ad-Luc versus Ad-Elovl6, Anova, one-way.

A: Immunoblot analysis for Elovl5. HepG2 cells were infected with Ad-Luc (control virus) or Ad-Elovl5 at 20 PFU/cells 48 hrs prior to treatment with [1-¹⁴C]-18:2, n-6 (Methods). The immunoblot represents the level of Elovl5 protein abundance in HepG2 following infection with Ad-Luc or Ad-Elovl5 prior to fatty acid treatment. Ad-Luc has no effect on Elovl5 protein abundance; Ad-Elovl5 infection increases Elovl5 protein 5-fold, a value consistent with physiological changes seen in vivo [24, 25]. B: Forty-eight hours after Ad-Luc or Ad-Elovl5 adenoviral infection, cells were treated with DMSO, soraphen A and [1-¹⁴C]-18:2,n-6 (50 µM) as described above. Six hours after treatment cells were harvested for extraction and saponification. ¹⁴C-fatty acids were fractionated as described (Methods). Treatments: Ad-Luc + DMSO (white bars); Ad-Elovl5 + DMSO (black bars); Ad-Elovl5 + soraphen A (gray bars). Results are expressed as % Distribution of ¹⁴C Fatty Acids; mean ± SD, n=3; *, p≤0.05 DMSO versus soraphen A; #, p≤0.05 Ad-Luc versus Ad-Elovl5; t-test.

HepG2 cells were infected recombinant adenovirus (Ad-Luc, Ad-Elovl5, Ad-Elovl6 [Panels A & B]; treated with DMSO or soraphen A (100 nM) and 50 µM ¹⁴C-palmitate [Panels A] or ¹⁴C-linoleate [Panels B & C] and as described in Figs. 3–6 above. The media from these studies was collected at the time of cell harvest and assayed for fatty acid oxidation products, ASM, acid soluble material (Methods). Results are reported as ASM, nmoles per mg protein, mean ± SD, n=3; *. p≤0.05 DMSO versus soraphen A; Anova, one-way.

HepG2 cells were infected Ad-Luc or Ad-FADS1 + Ad-FADS2 and treated with DMSO or soraphen A (100 nM) and 50 µM ¹⁴C-linoleate as described in Figs. 6. The media was collected at the time of cell harvest and assayed for fatty acid oxidation products, ASM, acid soluble material (Methods). Results are reported as ASM, nmoles/mg protein, mean ± SD, n=4; *. p≤0.05 DMSO versus soraphen A; #, Ad-Luc versus Ad-FADS1 + Ad-FADS2, Anova-one way.

See text for explanation.