A Mixture of Atropisomers Enhances Neutral Lipid Degradation in Mammalian Cells with Autophagy Induction

Abstract

Atropisomers with a biaryl dihydronaphthopyranone structure, dinapinones A1 (DPA1) (M position) and A2 (DPA2) (P position), were isolated from the fungus culture broth of Talaromyces pinophilus FKI-3864 as inhibitors of [¹⁴C]neutral lipid ([¹⁴C]triacylglycerol (TG) and [¹⁴C]cholesteryl ester (CE)) synthesis from [¹⁴C]oleic acid in Chinese hamster ovary-K1 (CHO-K1) cells. DPA2 inhibited [¹⁴C]TG and [¹⁴C]CE synthesis (IC₅₀s_, 0.65 and 5.6 μM, respectively), but DPA1 had no inhibitory activity on [¹⁴C]TG and [¹⁴C]CE synthesis even at 12 μM. However, a 1:1 mixture of DPA1 and DPA2 (DPA_mix) had the most potent inhibitory activity on [¹⁴C]TG and [¹⁴C]CE synthesis (IC₅₀s, 0.054 and 0.18 μM, respectively). The mechanism of action of DPA_mix was investigated. DPA_mix had no effects on the enzymes involved in neutral lipid synthesis, while DPA_mix enhanced the degradation of [¹⁴C]neutral lipids with concomitant decrease in cytosolic lipid droplets accumulated in CHO-K1 cells. From analysis of autophagy marker proteins, DPA_mix caused dose-dependent induction of microtubule-associated protein light chain 3-II (LC3-II) and degradation of p62. In the autophagic flux assay using bafilomycin A₁, DPA_mix upregulated autophagosome turnover. These results reveal that DPA_mix enhances neutral lipid degradation together with induction of autophagy.

Figure 1

Structures of dinapinones. Dinapinones A1 and A2 are homodimers, and the others are heterodimers.

Figure 2

Effects of DPA_mix on neutral lipid accumulation in three mammalian cells. After CHO-K1 cells (a), HeLa S3 cells (b) and HepG2 cells (c) were cultured with DPA_mix and [¹⁴C]oleic acid for 6 h, the cells were lysed and [¹⁴C]TG (■), [¹⁴C]CE (●) and [¹⁴C]PL (▲) were quantified by an image analyzer. The results obtained were plotted as % of control (without drugs). Values represent means ± SD (n = 3 ~ 4).

Figure 3

Effects of DPA_mix on neutral lipid degradation in CHO-K1 cells. (a,b) CHO-K1 cells were incubated for 24 h with [¹⁴C]oleic acid to accumulate [¹⁴C]lipids. After free [¹⁴C]oleic acid was removed, and cells were treated with DPA_mix for 0–12 h. (a) At indicated time, the cells treated with DPA_mix 0 (♦), 0.12 (▲), 1.2 (●) and 12 μM (■) were lysed and cellular [¹⁴C]TG (left) and [¹⁴C]CE (right) were detected by image analyzer. The results obtained were plotted as % of control (without drugs). Values represent means ± SD (n = 3 ~ 4). (b) At indicated time, the cells treated with DPA_mix 12 μM and the medium fractions were recovered to analyze the [¹⁴C]lipids in each fraction using image analyzer. The total amount of [¹⁴C]lipids in medium and cells is taken as 100% at each time. (c) CHO-K1 cells were cultured with 20 μM oleic acid. After the 12 h incubation, the lipid droplet-containing CHO-K1 cells (0 h) were incubated with DPA_mix for 6 h, and were stained with oil red O. Bars; 20 μm.

Figure 4

Effects of DPA_mix on autophagy flux in CHO-K1 cells. (a) After CHO-K1 cells were treated with DPA for 6 h, the cells were lysed and the cell lysates were analyzed by immunoblotting for LC3, p62 and β-actin. (b) After CHO-K1 cells were treated with DPA_mix (12 μM) for 1–6 h. At indicated time, the cells were lysed and the cell lysates were analyzed by immunoblotting for LC3, p62 and β-actin. (c) After CHO-K1 cells were treated with DPAB_mix, DPAC_mix, DPAD_mix or DPAE_mix for 6 h, the cells were lysed and the cell lysates were analyzed by immunoblotting for LC3, p62 and β-actin.