Anti-adipogenic and anti-obesity activities of purpurin in 3T3-L1 preadipocyte cells and in mice fed a high-fat diet

Abstract

Background: The body responds to overnutrition by converting stem cells to adipocytes. In vitro and in vivo studies have shown polyphenols and other natural compounds to be anti-adipogenic, presumably due in part to their antioxidant properties. Purpurin is a highly antioxidative anthraquinone and previous studies on anthraquinones have reported numerous biological activities in cells and animals. Anthraquinones have also been used to stimulate osteoblast differentiation, an inversely-related process to that of adipocyte differentiation. We propose that due to its high antioxidative properties, purpurin administration might attenuate adipogenesis in cells and in mice.

Methods: Our study will test the effect purpurin has on adipogenesis using both in vitro and in vivo models. The in vitro model consists of tracking with various biomarkers, the differentiation of pre-adipocyte to adipocytes in cell culture. The compound will then be tested in mice fed a high-fat diet. Murine 3T3-L1 preadipocyte cells were stimulated to differentiate in the presence or absence of purpurin. The following cellular parameters were measured: intracellular reactive oxygen species (ROS), membrane potential of the mitochondria, ATP production, activation of AMPK (adenosine 5'-monophosphate-activated protein kinase), insulin-induced lipid accumulation, triglyceride accumulation, and expression of PPARγ (peroxisome proliferator activated receptor-γ) and C/EBPα (CCAAT enhancer binding protein α). In vivo, mice were fed high fat diets supplemented with various levels of purpurin. Data collected from the animals included anthropometric data, glucose tolerance test results, and postmortem plasma glucose, lipid levels, and organ examinations.

Results: The administration of purpurin at 50 and 100 μM in 3T3-L1 cells, and at 40 and 80 mg/kg in mice proved to be a sensitive range: the lower concentrations affected several measured parameters, whereas at the higher doses purpurin consistently mitigated biomarkers associated with adipogenesis, and weight gain in mice. Purpurin appears to be an effective antiadipogenic compound.

Conclusion: The anthraquinone purpurin has potent in vitro anti-adipogenic effects in cells and in vivo anti-obesity effects in mice consuming a high-fat diet. Differentiation of 3T3-L1 cells was dose-dependently inhibited by purpurin, apparently by AMPK activation. Mice on a high-fat diet experienced a dose-dependent reduction in induced weight gain of up to 55%.

Keywords: Adipogenesis; Anthraquinone; Antioxidant; Cell differentiation; Lipogenic diet; Madder; Mitochondria; Obesity; Rubia cordifolia.

Fig. 1

Structure of purpurin

Fig. 2

Scheme of 3T3-L1 pre-adipocyte differentiation and purpurin treatment

Fig. 3

Cytotoxicity of purpurin on the murine 3T3-L1 pre-adipocyte cell line. Cells were cultured in the presence of purpurin (1, 10, 50, 100, and 250 μM) for 24 and 48 h. After treatment, cell survival was measured by the MTT assay. Data are expressed as the mean ± SD of triplicate experiments. Bars sharing a common letter are not significantly different at p < 0.05

Fig. 4

Effects of purpurin on intracellular ROS levels, mitochondrial membrane potential, and intracellular ATP levels in 3T3-L1 cells treated with MDI for 48 h: a cells were labeled with DCF-DA for cellular ROS detection, and JC-1 for the detection of mitochondrial membrane potential; b cells were lysed, centrifuged and the supernatant mixed with luciferase/luciferin reagent, after which free ATP was detected with a luminometer. Data are expressed as the mean ± SD of triplicate experiments. Bars sharing a common letter are not significantly different at p < 0.05

Fig. 5

Western Blot analysis of intracellular proteins from purpurin-treated cells in: a early-stage differentiation (2 d after MDI inducement), and b late-stage differentiation (8 d after MDI inducement). Cells were treated for the first 48 h with MDI and purpurin, after which these substances were removed from the culture. Figures represent results from at least three individual experiments. AMPK activity was expressed as the ratio phosphorylated to non-phosphorylated AMPK. Protein expression was calculated relative to β-actin, a constitutively expressed cellular protein

Fig. 6

Effect of purpurin on lipid accumulation in differentiated 3T3-L1 cells as measured with Oil Red O staining. The accumulation of lipid was evaluated: a microscopically; and (b) colorimetrically (absorbance at 520 nm) from solubilized fat droplets collected from the stained cells. Data are expressed as the mean ± SD of triplicate experiments. Bars sharing a common letter are not significantly different at p < 0.05

Fig. 7

Effect of purpurin on triglyceride deposition in fully differentiated 3T3-L1 cells. Data are expressed as the mean ± SD of triplicate experiments. Bars sharing a common letter are not significantly different at p < 0.05

Fig. 8

Body weight changes of male C57BL/6 mice fed a high-fat diet only (HFD), 40 mg/kg purpurin-contained high fat diet (HFD 40), or 80 mg/kg purpurin-contained high fat diet (HFD 80) for 10 weeks. # p < 0.5, ## p < 0.01, and ### p < 0.001 on means of HFD 40 versus high-fat diet. * p < 0.5, ** p < 0.01, and *** p < 0.001 on means of HFD 80 versus high-fat diet

Fig. 9

Effect of purpurin on glucose tolerance in high-fat diet fed mice. # p < 0.5, ## p < 0.01 on means of HFD 40 versus high-fat diet. * p < 0.5, ** p < 0.01 on means of HFD 80 versus high-fat diet

Fig. 10

Histological changes of liver (a) and white adipose tissue (b) in mice fed a high-fat diet or a purpurin-containing high-fat diet. Each tissue was fixed with 4% paraformaldehyde, and sections were stained with hematoxylin and eosin Y (H&E). Figures represent results from at least three separate experiments