A Gedunin-Type Limonoid, 7-Deacetoxy-7-Oxogedunin, from Andiroba (Carapa guianensis Aublet) Reduced Intracellular Triglyceride Content and Enhanced Autophagy in HepG2 Cells - PubMed (original) (raw)

. 2022 Oct 28;23(21):13141.

doi: 10.3390/ijms232113141.

Kiyofumi Ninomiya 1 2, Shinsuke Marumoto 3, Chie Sakai 1, Shuta Watanabe 1, Wakana Ishikawa 1, Yoshiaki Manse 1, Takashi Kikuchi 4 5, Takeshi Yamada 4, Reiko Tanaka 4, Osamu Muraoka 1 6, Toshio Morikawa 1 6

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A Gedunin-Type Limonoid, 7-Deacetoxy-7-Oxogedunin, from Andiroba (Carapa guianensis Aublet) Reduced Intracellular Triglyceride Content and Enhanced Autophagy in HepG2 Cells

Akifumi Nagatomo et al. Int J Mol Sci. 2022.

Abstract

The seed oil of Carapa guianensis Aublet (Andiroba) has been used in folk medicine for its insect-repelling, anti-inflammatory, and anti-malarial activities. This study aimed to examine the triglyceride (TG) reducing effects of _C. guianensis_-derived limonoids or other commercially available limonoids in human hepatoblastoma HepG2 cells and evaluate the expression of lipid metabolism or autophagy-related proteins by treatment with 7-deacetoxy-7-oxogedunin (DAOG; 1), a principal limonoid of C. guianensis. The gedunin-type limonoids, such as DAOG (% of control at 20 μM: 70.9 ± 0.9%), gedunin (2, 74.0 ± 1.1%), epoxyazadiradione (4, 73.4 ± 2.0%), 17β-hydroxyazadiradione (5, 79.9 ± 0.6%), 7-deacetoxy-7α-hydroxygedunin (6, 61.0 ± 1.2%), andirolide H (7, 87.4 ± 2.2%), and 6α-hydroxygedunin (8, 84.5 ± 1.1%), were observed to reduce the TG content at lower concentrations than berberine chloride (BBR, a positive control, 84.1 ± 0.3% at 30 μM) in HepG2 cells pretreated with high glucose and oleic acid. Andirobin-, obacunol-, nimbin-, and salannin-type limonoids showed no effect on the intracellular TG content in HepG2 cells. The TG-reducing effect of DAOG was attenuated by the concomitant use of compound C (dorsomorphin), an AMPK inhibitor. Further investigation on the detailed mechanism of action of DAOG at non-cytotoxic concentrations revealed that the expressions of autophagy-related proteins, LC3 and p62, were upregulated by treatment with DAOG. These findings suggested that gedunin-type limonoids from Andiroba could ameliorate fatty liver, and that the action of DAOG in particular is mediated by autophagy.

Keywords: Andiroba; Carapa guianensis; autophagy; fatty liver; gedunin; limonoid.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

Figure 1

Chemical structures of limonoids.

Figure 2

Figure 2

Oil red O staining of lipid droplets in HepG2 cells. Cells in the normal panel were treated with low glucose Dulbecco’s Modified Eagle Medium (DMEM) for 24 h. Reddish-colored particles are triglycerides accumulated lipid droplets in cells treated with high glucose and oleic acid. Sample or vehicle was applied for 24 h after induction of lipid accumulation.

Figure 3

Figure 3

Effect of DAOG (1) and BBR on HepG2 cell viability by MTT assay. 7-Deacetoxy-7-oxogedunin (DAOG; 1) and berberine chloride (BBR) were applied for 24 h after induction of intracellular triglyceride (TG) accumulation using high glucose and oleic acid. Each bar represents the mean with standard error of the mean (S.E.M.) (n = 4). Significantly different from the control, ** p < 0.01 (Dunnett).

Figure 4

Figure 4

Investigation of AMPK involvement in the mechanism of intracellular TG reduction in HepG2 cells. 7-Deacetoxy-7-oxogedunin (DAOG; 1) and berberine chloride (BBR) were applied for 24 h after induction of intracellular triglyceride (TG) accumulation by high glucose and oleic acid treatment. (A) Compound 1 and BBR reduced intracellular TG content in a concentration-dependent manner. The effects were abolished in the presence of a coexisting compound C (dorsomorphin); (B,C) Phosphorylation level of AMPK and acetyl-CoA carboxylase (ACC) in HepG2 cells treated with compound 1 (20 μM) or BBR (30 μM), as determined by western blot analysis. Compound C was used at a concentration of 20 μM. Unedited blots are shown in Figure S1. Each bar represents the mean with standard error of the mean (S.E.M.) (n = 4); * p < 0.05, ** p < 0.01 vs. control cells treated with vehicle (Dunnett); # p < 0.05, ## p < 0.01 vs. compound C-nontreated cells (Student’s t).

Figure 5

Figure 5

Expression of autophagy markers in HepG2 cells pretreated with high glucose and oleic acid concentrations. (A,B) Time-dependent expression of microtubule-associated protein 1 light chain 3 (LC3), sequestosome 1 (SQSTM1/p62), and Run domain Beclin-1 interacting and cysteine-rich containing protein (Rubicon) after application of 7-Deacetoxy-7-oxogedunin (DAOG; 1) and berberine chloride (BBR) in high glucose and oleic acid-pretreated HepG2 cells, determined by western blot analysis. Unedited blots are shown in Figure S2. Each bar represents the mean with standard error of the mean (S.E.M.) (n = 4); * p < 0.05, ** p < 0.01 vs. initial (0 h) expression (Dunnett); # p < 0.05, ## p < 0.01 vs. control group at each sampling point (Dunnett); (C) Autophagy flux assay for compound 1 and BBR with or without chloroquine (CQ; 100 μM). CQ was added 6 h before harvesting cells. Each bar represents the mean with S.E.M. (n = 4); different letters indicate significant differences, p < 0.05 (Tukey–Kramer’s HSD); (D) mRNA expression levels of p62 and Rubicon analyzed by quantitative reverse transcription polymerase chain reaction, standardized using RPLP0. Cells were collected 4 h after the application of samples for mRNA extraction. Each bar represents the mean with S.E.M. (n = 4); ** p < 0.01 vs. control group (Student’s t).

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