Aralia cordata inhibits triacylglycerol biosynthesis in HepG2 cells

Abstract

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the first committed step in triacylglycerol (TAG) and phospholipid biosynthesis, and has been considered as one of the drug targets for treating hepatic steatosis, insulin resistance, and other metabolic disorders. The aim of this study was to investigate the GPAT inhibitors from natural products and to evaluate their effects. The methanol extract of Aralia cordata roots showed a strong inhibitory effect on the human GPAT1 activity. A further bioactivity-guided approach led to the isolation of ent-pimara-8(14),15-dien-19-oic acid, (PA), one of the major compounds of A. cordata, which suppressed the GPAT1 activity with IC50 value of 60.5 μM. PA markedly reduced de novo lysophosphatidic acid synthesis through inhibition of GPAT activity and therefore significantly decreased synthesis of TAG in the HepG2 cells. These results suggest that PA as well as A. cordata root extract could be beneficial in controlling lipid metabolism.

FIG. 1.

Effect of MeOH extract from Aralia cordata and its CHCl₃ and H₂O phases on GPAT1 activity and de novo triacylglycerol (TAG) synthesis. (A) The MeOH extract and its partitions were tested against in vitro GPAT1 activity. (B) MTT assay was performed with increasing concentrations up to 30 μM for 24 h. (C, D) Lipid profile analyzed by TLC using either [¹⁴C] acetate or [¹⁴C] glycerol as radiolabeled substrates. Relative percentage of vehicle control represents was shown as a graph. Similar results were obtained in more than three independent experiments. *P<.05, **P<.01, ***P<.001 vs. vehicle control. AC-M, crude MeOH extract; AC-C, CHCl₃-soluble part separated out of AC-M; DMSO, dimethyl sulfoxide; BBR, berberine (positive control).

FIG. 2.

Activity-guided isolation of ent-pimara-8(14),15-dien-19-oic acid (PA) from the roots of A. cordata and its hGPAT1-inhibitory activity. (A) CHCl₃ partitions were sequentially fractionated into 11 subfractions using a stepwise gradient of hexane: EtOAc (10:1–1:1) and individual fractions analyzed for their effects on GPAT1 activity. Pure active compound (PA) was isolated using a silica column chromatography and chemical structure was shown below of schematic representation. (B) In vitro GPAT1 inhibitory activity of PA. Mitochondrial fractions of Sf-9 cells expressing hGPAT1 were prepared as described in Materials and Methods and the effects of PA on hGPAT1 activity with increasing concentrations were performed. Values are means (±standard deviation [SD]) for three separate preparations.

FIG. 3.

Effect of PA on lysophosphatidic acid (LPA) formation in HepG2 cells. (A) Lipid profile analyzed by TLC using [¹⁴C] glycerol as radiolabeled substrates. (B) The radioactivity of LPA bands were quantified by densitometry using the Multi-Gauge V3 program (Fuji Photo Film Co.) and relative percentage of vehicle control represents was shown as a graph. Data represent mean (±SD) obtained from three independent experiments. **P<.01, ***P<.001 versus vehicle control. STD, standard (¹⁴C-labeled LPA).

FIG. 4.

Effect of PA on de novo intracellular TAG synthesis in HepG2 cells. Lipid profile analyzed by TLC using either [¹⁴C] acetate (A), [¹⁴C] glycerol (B), or [¹⁴C] oleate (C) as radiolabeled substrates. Relative percentage of vehicle control represents was shown as a graph. Similar results were obtained in more than three independent experiments. Data represent mean (±SD) obtained from three independent experiments. *P<.05, **P<.01, versus vehicle control.