Anthocyanin-Enriched Riceberry Rice Extract Inhibits Cell Proliferation and Adipogenesis in 3T3-L1 Preadipocytes by Downregulating Adipogenic Transcription Factors and Their Targeting Genes

Figures

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  Figure 1. Schematic representation of 3T3-L1 differentiation into adipocytes.

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  Figure 2. Effect of Riceberry rice extract (RBE) on 3T3-L1 preadipocyte proliferation during a 4-day period. (A) Treatment of cells with RBE did not affect cell viability. (B) RBE at 20 µg/mL significantly decreased the total cell number. Bright-field images (20× magnification) show the reduction in preadipocyte under RBE treatment compared to the control group. (C) RBE at 10 and 20 µg/mL induced cell cycle arrest with increased G0/G1 and decreased G2/M phases. The results are expressed as mean ± SEM from the three independent experiments. p < 0.05 for groups with different letters. Scale bars are 100 µm.

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  Figure 3. Effect of Riceberry rice extract (RBE) on intracellular calcium signals in 3T3-L1 cells. (A) Stimulation of cells with RBE 1, 10, and 20 µg/mL concentrations failed to induce calcium signals compared to 1 µM ionomycin as a positive control. Black lines represent average traces from all cells. Grey lines represent individual cell recordings. (B) Fluorescence emission from cells prior to, during RBE, and ionomycin treatments. Scale bars are 100 µm.

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  Figure 4. Effect of Riceberry rice extract (RBE) on adipogenesis in 3T3-L1 cells during an 8-day period. (A) Treatment of cells with RBE did not affect cell viability during adipogenesis. (B) RBE at 20 µg/mL significantly decreased the total adipocyte number. (C) RBE (1–20 µg/mL) decreased adipocyte number in a concentration-dependent manner. (D) A significant reduction in triglyceride accumulation was observed in mature adipocytes. (E) Treatment of cells with RBE inhibited adipogenesis by decreasing adipocyte number, cell size, and lipid droplet content as shown by Oil Red O staining (20× magnification). The results are expressed as mean ± SEM from the three independent experiments. Groups with a different letter represent statistical significance (p < 0.05). U: undifferentiated cells; D: differentiated cells. Scale bars are 100 µm.

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  Figure 5. Effect of Riceberry rice extract (RBE) on adipogenic transcription factors and Akt1 signaling in 3T3-L1 cells. (A) RBE (20 µg/mL) significantly downregulated mRNA expression of PPARγ, C/EBPα, and C/EBPβ, and upregulated C/EBPγ in differentiated cells. (B) Treatment of cells with RBE tended to reduce the relative values of phospho-Akt1 (S473) to total-Akt1 in differentiated cells. The results are expressed as mean ± SEM from the three independent experiments. Groups with a different letter represent statistical significance (p < 0.05). Data were normalized with β-actin as an internal control. U: undifferentiated cells; D: differentiated cells.

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  Figure 6. Effect of Riceberry rice extract (RBE) on adipogenic gene mRNA expression. Treatment with RBE (20 µg/mL) significantly downregulated adipogenic gene mRNA expression for ACC, aP2, Leptin, AdipoQ, AdipoQ-R1, AdipoQ-R2, Resistin, ATGL, HSL, LPL, and perilipin in differentiated cells. The results are expressed as mean ± SEM from the three independent experiments. Groups with a different letter represent statistical significance (p < 0.05). Data were normalized with β-actin as an internal control. U: undifferentiated cells; D: differentiated cells.

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  Figure 7. Effect of Riceberry rice extract (RBE) on glucose uptake in mature adipocytes. (A) RBE at 10 and 20 µg/mL significantly reduced glucose uptake into mature adipocytes after 8 days of treatment. (B) Downregulation of Glut4 mRNA expression in response to RBE at 20 µg/mL in differentiated cells. (C) Treatment of cells with RBE did not impact insulin receptor (InsR) mRNA expression. The results are expressed as mean ± SEM from the three independent experiments. Groups with a different letter represent statistical significance (p < 0.05). Data were normalized with β-actin as an internal control. U: undifferentiated cells; D: differentiated cells.

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  Figure 8. Effect of Riceberry rice extract (RBE) on lipolysis in adipocytes. RBE (20 µg/mL) significantly enhanced isoproterenol-induced glycerol release from adipocytes. The results are expressed as mean ± SEM from the three independent experiments. Groups with a different letter represent statistical significance (p < 0.05).

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  Figure 9. Schematic summary of the inhibitory effect of Riceberry rice extract (RBE) on cell proliferation and differentiation in 3T3-L1 adipocytes. RBE suppressed preadipocyte proliferation through inducing cell cycle arrest. RBE downregulated adipogenic transcription factors and their target genes, leading to inhibit adipogenesis and reduce glucose uptake. In addition, RBE enhanced isoproterenol-induced lipolysis in mature adipocytes.