Rubus crataegifolius Bunge regulates adipogenesis through Akt and inhibits high-fat diet-induced obesity in rats - PubMed (original) (raw)
Rubus crataegifolius Bunge regulates adipogenesis through Akt and inhibits high-fat diet-induced obesity in rats
Min-Sup Jung et al. Nutr Metab (Lond). 2016.
Abstract
Background: Obesity is one of the greatest public health problems and major risk factors for serious metabolic diseases and significantly increases the risk of premature death. The aim of this study was to determine the inhibitory effects of Rubus crataegifolius Bunge (RCB) on adipocyte differentiation in 3 T3-L1 cells and its anti-obesity properties in high fat diet (HFD)-induced obese rats.
Methods: 3 T3-L1 adipocytes and HFD-induced obese rats were treated with RCB, and its effect on gene expression was analyzed using RT-PCR and Western blotting experiments.
Results: RCB treatment significantly inhibited adipocyte differentiation by suppressing the expression of C/EBPβ, C/EBPα, and PPARγ in the 3 T3-L1 adipocytes. Subsequently, the expression of the PPARγ target genes aP2 and fatty acid synthase (FAS) decreased following RCB treatment during adipocyte differentiation. In uncovering the specific mechanism that mediates the effects of RCB, we demonstrated that the insulin-stimulated phosphorylation of Akt strongly decreased and that its downstream substrate phospho-GSK3β was downregulated following RCB treatment in the 3 T3-L1 adipocytes. Moreover, LY294002, an inhibitor of Akt phosphorylation, exerted stronger inhibitory effects on RCB-mediated suppression of adipocyte differentiation, leading to the inhibition of adipocyte differentiation through the downregulation of Akt signaling. An HFD-induced obesity rat model was used to determine the inhibitory effects of RCB on obesity. Body weight gain and fat accumulation in adipose tissue were significantly reduced by the supplementation of RCB. Moreover, RCB treatment caused a significant decrease in adipocyte size, associated with a decrease in epididymal fat weight. The serum total cholesterol (TC) and triglyceride (TG) levels decreased in response to RCB treatment, whereas HDL cholesterol (HDL-C) increased, indicating that RCB attenuated lipid accumulation in adipose tissue in HFD-induced obese rats.
Conclusion: Our results demonstrate an inhibitory effect of RCB on adipogenesis through the reduction of the adipogenic factors PPARγ, C/EBPα, and phospho-Akt. RCB had a potent anti-obesity effect, reducing body weight gain in HFD-induced obese rats.
Keywords: 3 T3-L1 adipocyte; Adipogenesis; Akt; High fat diet; Obesity; Rubus crataegifolius Bunge (RCB).
Figures
Fig. 1
Effect of RCB on adipocyte differentiation in 3 T3-L1 cells. a Lipid accumulation in 3 T3-L1 adipocytes assessed by Oil red O staining. 3 T3-L1 pre-adipocytes were differentiated into mature adipocytes and then treated with RCB for 7 days. At day 7 post-induction, the cells were fixed, and neutral lipids were stained with Oil red O. DMI, fully differentiated adipocytes (0.5 mM 3 IBMX, 100 μM indomethason, 0.25 μM dexamethasone and 167 nM insulin); 50, fully differentiated adipocytes (DMI + 50 μg/ml RCB); 150, fully differentiated adipocytes (DMI + 150 μg/ml RCB). RCB represents Rubus crataegifolius Bunge extracts. The scale bar is 50 μm. b Effect of RCB on TG accumulation in differentiating 3 T3-L1 cells. Triglyceride content was measured using a triglyceride assay kit. The results shown are representative of at least three independent experiments. The values are presented as the means ± SD. Different letters on the each bar graph indicate that the differences were statically significant (I 0.05) according to Duncan’s multiple range test. c Effect of RCB on cytotoxicity during 3T3L1 pre-adipocyte differentiation into adipocytes. 3 T3-L1 cells were treated with RCB at various concentrations (0, 50, or 150 μg/ml) in a DMI mixture for 4 or 7 days. Cell viability after treatment with RCB was determined with an MTT assay
Fig. 2
Effects of RCB on the mRNA and protein expression of genes related to adipogenesis. a RCB inhibited the mRNA expression of adipocyte-specific factors during 3 T3-L1 adipocyte differentiation. Differentiation of 3 T3-L1 pre-adipocytes into adipocytes was induced using DMI media in the absence or presence of RCB (50 μg/mL or 150 μg/mL) for 5 days. β-actin expression in each sample was used as an internal control to normalize expression. The results shown are representative of at least three independent experiments. The different letters on each bar graph indicate significant differences (p < 0.05), as determined by Duncan’s multiple range test. b RCB inhibited the protein expression of adipocyte-related factors in 3 T3-L1 adipocytes. Cell lysates from the 3 T3-L1 cells were prepared at day 4 or day 7 after the induction of differentiation. Western blotting analysis was described in detail in the Materials and Methods section
Fig. 3
Effect of RCB on Akt and GSK3β phosphorylation during 3 T3-L1 adipogenesis. a RCB downregulated Akt phosphorylation induced by DMI in 3 T3-L1 adipocytes. The 3 T3-L1 pre-adipocytes were induced to differentiate with DMI media in the absence or presence of RCB for 4 or 7 days, and the phosphorylation levels of Akt were detected with its specific antibody. The results are reported as the means ± SD of three independent experiments. *P < 0.05. **P < 0.01. b Effect of RCB on GSK3β phosphorylation during 3 T3-L1 adipogenesis. The phosphorylation levels of GSK3β were determined using a specific antibody. The results are reported as the means ± SD of three independent experiments. *P < 0.05. c Inhibitory effects of LY294002 on 3 T3-L1 adipocyte differentiation. 3 T3-L1 cells were incubated with or without RCB at a concentration of 50 or 150 μg/ml during differentiation in the presence or absence of LY294002 (10 μM). After differentiation, the TG contents in the 3 T3-L1 adipocytes were determined by a triglyceride assay. The data presented are the mean ± SD from three independent experiments. *P < 0.05. d RCB inhibited DMI-stimulated glucose uptake in 3 T3-L1 adipocytes. Glucose uptake activity in differentiated 3 T3-L1 cells was analyzed by measuring the fluorescence intensity of the cells after incubation with 10 μM 2-NBDG for 2 h. The results are reported as the mean ± SD of three independent experiments. *P < 0.05. **P < 0.01
Fig. 4
Effects of RCB extracts on body weight in HFD-induced obese rats. a Body weight gain. The rats were divided into three groups (n = 10): an ND group given a normal diet (ND), an HFD group fed an HFD, and an HFD + RCB group fed an HFD in addition to treatment with RCB (200 mg/kg BW) orally by gavage once a day for 5 weeks. There were significant differences between the body weights of the HFD and ND (**P < 0.01) and HFD and HFD + RCB groups (*P < 0.05) at the end of the experimental period. b Food intake. The mean daily food consumption was 26.7 g and no significant differences were found in food intake between ND and HFD groups. c Adipose tissue mass. The weight of the epididymal adipose tissue was measured by dividing fatty tissue weight by body weight (fatty tissue/body weight x 100). Values represent the means ± SD; P < 0.05, as shown by ANOVA. Bars labeled with different letters indicate significant differences at according to Duncan’s multiple range test. d The weight of the perirenal adipose tissue was measured by dividing fatty tissue weight by body weight (fatty tissue/body weight x 100). The data presented are the mean ± SD from three independent experiments. Values represent the means ± SD; P < 0.05 as shown by ANOVA. Different letters (a and b) mean that values are significantly different among groups. e Representative hematoxylin and eosin-stained sections of epididymal adipose tissue. The adipocyte sizes from the HFD + RCB group were smaller than those from the HFD only group. The scale bar is 100 μm
Fig. 5
Effect of RCB on lipid content in HFD-induced obese rats. a Serum triglyceride level. The RCB extract-administered group showed significantly lower levels of serum triglyceride compared with the HFD group. Data are expressed as the means ± SD. Differences among multiple groups were analyzed with ANOVA followed by Duncan’s multiple range test. Different letters (a and b) indicate that the column means are significantly different at P < 0.05. b Total cholesterol level. Total cholesterol level decreased in the RCB-treated groups compared to the HFD only group. The results are presented as the means ± SD. Different letters (a and b) indicate that the column means are significantly different; p < 0.05 according to Duncan’s multiple range test. c HDL cholesterol level. A significant difference was observed in HDL cholesterol levels between the HFD and HFD + RCB groups. The values are expressed as the means ± SD. Different letters (a and b) on the each bar graph mean that values are significantly different among groups; p < 0.05 according to Duncan’s multiple range test
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