Genistin: A Novel Potent Anti-Adipogenic and Anti-Lipogenic Agent

Abstract

Soy isoflavones are popular ingredients with anti-adipogenic and anti-lipogenic properties. The anti-adipogenic and anti-lipogenic properties of genistein are well-known, but those of genistin and glycitein remain unknown, and those of daidzein are characterized by contrasting data. Therefore, the purpose of our study was to investigate the effects of daidzein, glycitein, genistein, and genistin on adipogenesis and lipogenesis in 3T3-L1 cells. Proliferation of 3T3-L1 preadipocytes was unaffected by genistin and glycitein, but it was affected by 50 and 100 µM genistein and 100 µM daidzein for 48 h. Among the four isoflavones, only 50 and 100 µM genistin and genistein markedly suppressed lipid accumulation during adipogenesis in 3T3-L1 cells through a similar signaling pathway in a dose-dependent manner. Genistin and genistein suppress adipocyte-specific proteins and genes, such as peroxisome proliferator-activated receptor γ (PPARγ), CCAAT-enhancer-binding protein α (C/EBPα), and adipocyte binding protein 2 (aP2)/fatty acid-binding protein 4 (FABP4), and lipogenic enzymes such as ATP citrate lyase (ACL), acetyl-CoA carboxylase 1 (ACC1), and fatty acid synthase (FAS). Both isoflavones also activate AMP-activated protein kinase α (AMPKα), an essential factor in adipocyte differentiation, and inhibited sterol regulatory element-binding transcription factor 1c (SREBP-1c). These results indicate that genistin is a potent anti-adipogenic and anti-lipogenic agent.

Keywords: anti-adipogenesis; anti-lipogenesis; genistin; soy isoflavones.

Scheme 1

Structure of the four soy isoflavones.

Figure 1

Effects of soy isoflavones on 3T3-L1 cell proliferation. 3T3-L1 preadipocytes were treated with 25, 50, and 100 μM daidzein, glycitein, genistein, and genistin for (A) 24 h and (B) 48 h, and cell proliferation was measured using CCK-8 assays. All data are presented as the mean ± SD and analyzed with one-way ANOVA and compared with those from the isoflavone-untreated control (n = 3, * p < 0.05, ** p < 0.005, *** p < 0.001). Live/dead cells were imaged to confirm the cytotoxicity of each compound. Green: live; red: dead; blue: nucleus.

Figure 2

Suppressive effect of genistin and genistein on adipocytic differentiation of 3T3-L1 cells. During differentiation, 3T3-L1 cells were separately treated with 25, 50, or 100 μM of each soy isoflavone in the MDI media (a mixture of 3-isobutyl-1-methylxanthine (M), dexamethasone (D), and insulin (I)). Lipid accumulation was determined by Oil Red O (ORO) staining. (A) Data from ORO staining of the four soy isoflavones were quantified. All data are presented as the mean ± SD and analyzed with one-way ANOVA and compared with those from the isoflavone-untreated 5control. (n ≥ 3, ** p < 0.005, *** p < 0.001). (B) ORO images of genistein- and genistin-treated 3T3-L1 cells were obtained. Pre, preadipocytes; con, adipocytes.

Figure 3

Effect of genistin and genistein on the protein expression and messenger RNA (mRNA) levels of adipogenic-specific factors in 3T3-L1 adipocytes. 3T3-L1 cells were completely differentiated in the MDI media in the presence of 25, 50, or 100 μM genistin or genistein during the differentiation process. (A) Protein expression changes of C/EBPα, PPARγ, and FABP4 in 3T3-L1 cells was monitored using Western blotting. (B–D) The mRNA expression of adipogenic factors, including (B) C/EBPα, (C) PPARγ, and (D) aP2, was calculated. All data were expressed as fold changes in expression relative to that in the control, untreated adipocytes (n ≥ 3, ^### p < 0.001 vs. Pre (preadipocyte) group; * p < 0.05, ** p < 0.005, *** p < 0.001 vs. Con (control, adipocyte) group; one-way ANOVA with the Tukey’s post-hoc test). C/EBPα, CCAAT-enhancer-binding protein α; PPARγ, peroxisome proliferator-activated receptor γ; FABP4, fatty acid-binding protein 4; aP2, adipocyte-binding protein 2.

Figure 4

Effect of genistin and genistein on mRNA levels of lipogenic-related factors in 3T3-L1 adipocytes. 3T3-L1 cells were completely differentiated in the MDI media in the presence of 25, 50, or 100 μM genistin or genistein during the differentiation process. The mRNA expression of lipogenic factors, including (A) ACL, (B) ACC1, and (C) FAS was evaluated. All data were expressed as fold changes in expression relative to that in the control, untreated adipocytes (n ≥ 3, ^### p < 0.001 vs. Pre (preadipocyte) group; * p < 0.05, *** p < 0.001 vs. Con (control, adipocyte) group; one-way ANOVA with the Tukey’s post-hoc test). ACL, ATP citrate lyase; ACC1, acetyl-CoA carboxylase 1; FAS, fatty acid synthase.

Figure 5

Effect of genistin and genistein on protein expression and mRNA levels of AMPKα and SREBP-1c in 3T3-L1 adipocytes. 3T3-L1 cells were completely differentiated in the MDI media in the presence of 25, 50, or 100 μM genistin or genistein during the differentiation process. (A) Protein expressions of AMPKα and phosphorylated AMPKα were investigated using Western blotting. (B) Gene expression of SREBP-1c was estimated. All data were expressed as fold changes in expression relative to that in the control, untreated adipocytes (n ≥ 3, ^### p < 0.001 vs. Pre (preadipocyte) group; * p < 0.05, *** p < 0.001 vs. Con (control, adipocyte) group; one-way ANOVA with the Tukey’s post-hoc test). AMPK, AMP-activated protein kinase; SREBP-1c, sterol regulatory element-binding transcription factor 1c.

Figure 6

Proposed mechanism of genistin in adipogenesis and lipogenesis.