(-)- O-Methylcubebin from Vitex trifolia Enhanced Adipogenesis in 3T3-L1 Cells via the Inhibition of ERK1/2 and p38MAPK Phosphorylation

Abstract

In this study, for the purpose of elucidation for antidiabetic components, we isolated and identified compounds that could become lead compounds for the development of antidiabetic agents from the herbal medicine Vitex trifolia, which is used for liver protection in Myanmar. Three kinds of lignan, (-)-O-methylcubebin (MC), (-)-hinokinin, and (-)-cubebin, were isolated from the ethyl acetate extract of the leaves of V. trifolia, using various chromatography. Among the three isolated compounds, MC showed the strongest effects to increase intracellular lipid accumulation in 3T3-L1 cells. From the results of the elucidation of the MC's effects on the adipogenesis of 3T3-L1 cells, the downsizing of adipocytes and the promotion of the expression of adipogenesis-related proteins, as well as adiponectin, were observed. On the other hand, since the activity of MC was inhibited by antagonists of PPARγ and improved by inhibitors of the classical mitogen-activated protein kinase (MAPK) pathway and p38MAPK pathway, MC was considered to be an agonist of PPARγ, and furthermore promoted adipogenesis via the inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38MAPK phosphorylation. Although MC showed similar effects to those of rosiglitazone (RO) used as a positive control, RO promoted the migration of GLUT4 from the cytoplasm to the cell membrane, whereas MC did not show such an effect. From the abovementioned results, it was considered that MC could be a lead compound for the development of antidiabetic drugs that does not show weight gain, which is a side effect of RO.

Keywords: (-)-O-methylcubebin; Vitex trifolia L.; adipogenesis; lipogenesis; lipolysis; rosiglitazone.

Figure 1

Isolated Compounds.

Figure 2

Cytotoxic effects of the three compounds isolated from V. trifolia, rosiglitazone (RO), and berberine (BER) in 3T3-L1 cells. B(Black): Cultrued cells without any compund. Data are expressed as the mean ± SD from three independent experiments. The same letters indicate that there are no differences between those groups, and different letters indicate significant differences (P < 0.05).

Figure 3

The effects of the compounds isoloated from V. trifolia on the liped levels in 3T3-L1 cells. B (Black): Undifferentiated cells without the addition of the MDI mixture [a mixture of 0.5 mM 3-isobuty-1-methyl xanthine (M), 0.1 µM dexamethasone (D) and 2 µM insulin (I)], C (Control): cells with the addition of the MDI, BER: cells with MDI and berberine (2.7 nM), and RO: cells with MDI and rosiglitazone (100 nM). The arrow indicated the addition of the MDI mixture. Data are presented as the mean ± SD from three independent experiments. The same letters indicate that there are no differences between those groups, and different letters indicate significant differences (P < 0.05).

Figure 4

Images and the diameter of 3T3-L1 cells on the say 8 with reference compounds or methylcubelin (MC) of various concentrations. (a) Images of 3T3-L1 cells treated with corresponding conditions; (b) Cell diameters were determined using Image J software. B (Black): Undifferentiated cells without the addition of the MDI mixture. C (Control): cells with the addition of the MDI Arrow (solid line) indicates the addition of the MDI mixture. Data are presented as the mean ± SD from 100 cells of three independent pictures. The same letters indicate that there are no differences between those groups, and different letters indicate significant differences (P < 0.05).

Figure 5

The effects of each compound on adipogenesis-related proteins, adiponectin, and GLUT4 levels in 3T3-L1 cells on day 8. Since all the same samples were used and meansurements were performed using 13 types of primary antibodies, only one β-actin image was shown. Protein levels were measured by electroblotting. B (Black): Undifferentiated cells without the addition of the MDI, C (Control): cells with the addition of the MDI, BER: cells with MDI and berberine (2.7 nM), and RO: cells with MDI and rosiglitazone (100 nM). The number with MC indicate the concentration (µM). Arrows indicate the addition of the MDI mixture. Data are presented as the mean ± SD from three independent experiments. The test results of the significance of protein expression levels are summarized in Table S2 of the Supplemetary Materials.

Figure 6

The effects of each compound on the intracellular signal transduction-related protein levels in 3T3-L1 cells on day 8. Protein levels were measured by electroblotting. B (Black): Undifferentiated cells without the addition of the MDI, C (Control): cells with the addition of the MDI, BER: cells with MDI and berberine (2.7 nM), and RO: cells with MDI and rosiglitazone (100 nM). The number with MC indicate the concentration (µM). Arrows indicate the addition of the MDI mixture. Data are presented as the mean ± SD from three independent experiments. The same letters indicate that there are no differences between those groups, and different letters indicate significant differences (P < 0.05).

Figure 7

The effects of each inhibitor on the lipid levels in 3T3-L1 cells during adipogenesis. B (Black): Undifferentiated cells without the addition of the MDI, PPAR: cells with MDI and bisphenol A diglycidyl ether, AKt: cells with MDI and wortmannin, MEK: cells with MDI and U0126, p38: cells with MDI and SB202190, and JNK: cells with MDI and JNK inhibitor. The data are presented as the mean ± SD from three independent experiments. The arrow indicates the addition of the MDI mixture. The same letters indicate that there are no differences between those groups, and different letters indicate significant differences (P < 0.05).