Genistein and daidzein repress adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells via Wnt/β-catenin signalling or lipolysis

Abstract

Objectives: One aspect of the effects of isoflavones against fat deposition might be at least associated with the mechanism by which Wnt/β-catenin signalling inhibits adipocyte differentiation. However, it remains completely unknown as to whether isoflavones might influence Wnt signalling during commitment of pluripotent mesenchymal stem cells (MSCs) to adipose lineages. In the present study, we have investigated the mechanisms underlying effects of genistein and daidzein, the major soy isoflavones, on anti-adipogenic Wnt/β-catenin signalling.

Materials and methods: Adipose tissue-derived (AD) MSCs were exposed continuously to genistein and daidzein (0.01-100 μm) during adipogenic differentiation (21 days). An oestrogen antagonist, ICI 182,780, was used to determine whether or not the isoflavones activated Wnt signalling via oestrogen receptors (ERs).

Results: Genistein and daidzein suppressed adipogenic differentiation of AD-MSCs in a dose-dependent manner and inhibited expression of adipogenic markers, PPARγ, SREBP-1c and Glut 4, from mid-phase differentiation. Microarrays showed that anti-adipogenic effects of genistein were principally attributable to activation of Wnt signalling via ERs-dependent pathway, such as Erk/JNK signalling and LEF/TCF4 co-activators. These findings were supported by evidence that the effects of genistein were offset by ICI182,780. Unlike genistein, daidzein inhibited adipogenesis through stimulation of lipolysis, with for example, PKA-mediated hormone sensitive lipase. This is consistent with the increase in glycerol released from AD-MSCs. In conclusion, understanding that different sets of mechanisms of the two isoflavones on adipogenesis will help the design of novel strategies to prevent observed current epidemic levels of obesity, using isoflavones.

Figure 1

Genistein and daidzein inhibited adipogenic differentiation of AD‐MSCs in a dose‐dependent manner. AD‐MSCs were cultured in adipogenic differentiation medium plus genistein or daidzein for 21 days. (A) oil red O staining (original magnification; 200×, scale bar; 100 μm); (B) Quantification of oil red O; (C) Hoechst staining (original magnification; 100×); (D) MTT assay at 1, 2 and 3 days; (E) Protein level of apoptotic Bax and anti‐apoptotic Bcl‐2; (F) mRNA level of adipogenic PPARγ and anti‐adipogenic β‐catenin. Data are expressed as mean ± SEM (n = 3–5 independent experiments). *Mean values were significantly different from those of control groups (Dunnett’s test; P < 0.05). AD‐MSCs, adipose tissue‐derived mesenchymal stem cells; PPARγ, peroxisome proliferator‐activated receptor γ.

Figure 2

Genistein and daidzein inhibited adipogenic differentiation of AD‐MSCs from mid‐phase differentiation. AD‐MSCs were cultured in adipogenic differentiation medium plus genistein or daidzein for 3 (early), 12 (middle) and 21 (terminal) days. (A) oil red O (original magnification; 200×, scale bar; 100 μm) and Hoechst staining (original magnification; 100×); (B) Quantification of oil red O; (C) Immunofluorescence of Oct‐4 (original magnification; 400×, scale bar; 100 μm). Data are expressed as mean ± SEM (n = 3–5 independent experiments). *Mean values were significantly different from those of control groups (Dunnett’s test; P < 0.05). AD‐MSCs, adipose tissue‐derived mesenchymal stem cells.

Figure 3

Genistein and daidzein altered expression of adipogenic or anti‐adipogenic markers principally from mid‐phase. AD‐MSCs were cultured in adipogenic differentiation medium plus genistein or daidzein for 3 (early), 12 (middle) and 21 (terminal) days. (A) mRNA levels of anti‐adipogenic genes; (B) mRNA level of adipogenic genes; (C) Protein levels of β‐catenin and PPARγ. Data are presented as mean ± SEM (n = 3–5 independent experiments). *Mean values were significantly different from those of control groups at each differentiation phase (3, 12 and 21 days) (Dunnett’s test; P < 0.05). AD‐MSCs, adipose tissue‐derived mesenchymal stem cells; aP2, fatty acid‐binding protein 4; Glut 4, glucose transporter 4; PPARγ, peroxisome proliferator‐activated receptor γ; SREBP‐1c, sterol regulatory element‐binding protein‐1c.

Figure 4

Inhibitory effects of genistein and daidzein on adipogenesis were exerted via ER‐dependent mechanism. AD‐MSCs were cultured in adipogenic differentiation medium plus genistein or daidzein or combination with ICI for 3 (early), 12 (middle) and 21 (terminal) days. (A) oil red O (original magnification; 200×, scale bar; 100 μm) and Hoechst staining (original magnification; 100×); (B) Quantification of oil red O; (C) Protein level of ER signalling‐related intermediates, β‐catenin and PPARγ; (D) Cellular triglyceride and glycerol concentration. Data are expressed as mean ± SEM (n = 3–5 independent experiments). *Mean values were significantly different from those of control groups at each differentiation phase (3, 12 and 21 days) (Dunett’s test; P < 0.05), ^†mean values were significantly different between groups with ICI and without ICI addition (t‐test; P < 0.05). ^a,b,cMean values with dissimilar superscript letters were significantly different between each groups (Duncan’s test; P < 0.05).