Allyl isothiocyanate suppresses the proteolytic activation of sterol regulatory element-binding proteins and de novo fatty acid and cholesterol synthesis

Abbreviations: ACC1, acetyl-CoA carboxylase 1; AITC, allyl isothiocyanate; AMPK, AMP-activated kinase; COP II, coated protein II; DMEM, Dulbecco’s modified Eagle’s medium; ER, endoplasmic reticulum; FAS, fatty acid synthase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; 25-HC, 25-Hydroxycholesterol; 4′-HF, 4′-hydroxyflavanone; HMGCR, HMG-CoA reductase; HMGCS, HMG-CoA synthase; Insigs, insulin-induced genes; LPDS, lipoprotein-deficient serum; SCAP, SREBP cleavage-activating protein; SCD1, stearoyl-CoA desaturase 1; S1P, site-1 protease; S2P, site-2 protease; SQS, squalene synthase; SREBPs, sterol regulatory element-binding proteins; XN, xanthohumol.

Figures & data

Fig. 1. AITC reduces the activity of the SRE-containing FAS promoter.

Notes: (A) Sterols in Huh-7/FAS cells were depleted by incubating these cells in medium B for 16 h. The cells were then transferred to medium B in the presence of vehicle, 2.5 μM 25-HC, 10 μM AITC, 30 μM AITC, or 100 μM AITC. After incubating for 24 h, the luciferase assay was performed and the relative luciferase activities were obtained by normalizing with respect to the protein content in the cell extracts. Luciferase activity in the presence of the vehicle is represented as 1.0. (B) Structure of AITC. All data are presented as mean ± S.E. (n = 3). Different superscript letters denote statistical significance (p < 0.05).

Fig. 2. AITC suppresses the processing of SREBP-1 and SREBP-2. Sterols in Huh-7 cells were depleted by incubating these cells in medium C for 16 h.

Notes: The cells were then transferred to medium C in the presence of vehicle, 2.5 μM 25-HC, 10 μM AITC, 30 μM AITC, or 100 μM AITC. After incubating for 3 h, whole-cell extracts from the cells were subjected to immunoblotting with anti-SREBP-1, anti-SREBP-2, or anti-β-actin antibodies. The same results were obtained in more than three separate experiments.

Fig. 3. AITC suppresses SREBP target gene expression as well as the de novo synthesis of fatty acids and cholesterol.

Notes: (A) Sterols in Huh-7 cells were depleted by incubating these cells in medium C for 16 h. The cells were then transferred to medium C in the presence of vehicle or 100 μM AITC. After incubating for 24 h, total RNA was isolated from the cells. Real-time quantitative PCR was performed, and the relative mRNA levels of the indicated genes were normalized with respect to those of GAPDH mRNA. The mRNA levels of various genes in the presence of vehicle are represented as 1.0. (B) Huh-7 cells were cultured in medium D for 16 h. The cells were then transferred to medium D in the presence of vehicle or 100 μM AITC. After incubating for 18 h, the cells were treated with 1.6 μCi/mL [¹⁴C]-acetate and cultured for an additional 6 h. Fatty acids and cholesterol were extracted, and the incorporation of [¹⁴C]-acetate into them was determined. All data are presented as mean ± S.E. (n = 3). *p < 0.05, **p < 0.01.