Measurement of total phenolic content using folin-ciocalteu assay

The total phenolic content of RCB was measured using a spectrophotometer according to the Folin-Ciocalteu colorimetric method as previously described [21, 22]. Because quercetin is one of the polyphenol compounds found in RCB, the total phenolic content of the ethanol extract of RCB was expressed as mg catechin equivalents (QE)/g. Catechin was purchased from Sigma-Aldrich (St. Louis, MO. USA). The measurements were performed four times.

Measurement of total flavonoids

Total flavonoid content was determined as previously described [23] with slight modifications. Briefly, 0.25 mL of RCB (100 μg/mL) was added to a tube containing 1 mL of double-distilled water. Following this, 0.075 mL of 5 % NaNO₂, 0.075 mL of 10 % AlCl₃ and 0.5 mL of 1 M NaOH were added sequentially at 0, 5 and 6 min. Finally, the volume of the reacting solution was adjusted to 2.5 mL with double-distilled water. The 410 nm absorbance of the solution was detected using an Ultrospec 2100 Pro Spectrophotometer (Section 3.3). The results are expressed in mg quercetin equivalents (QE)/g. The experiments were carried out in quadruplicate.

Measurement of free radical scavenging activity using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay

The free radical scavenging activity of RCB (100 μg/mL in DW) was measured using a method created by Brand-Williams [24] with slight modifications. The inhibition percentage was calculated using the following equation: Inhibition %=[(absorbance of control - absorbance of sample)/absorbance of control]×100. The absorbance was measured using a spectrophotometer (Ultrospec 2100 pro; Amersham Pharmacia Biotech Co., Piscataway, NJ, USA). The experiments were carried out in quadruplicate.

Measurement of superoxide anion (O2^●−) radical scavenging and hydroxyl (OH^●) radical scavenging activity

Superoxide radicals were generated according to a method described in a previous paper [25]. The samples (100 μg/mL in DMSO) were added to a reaction solution containing 100 μL of 30 mM EDTA (pH 7.4), 10 μL of 30 mM hypoxanthine in 50 mM NaOH, and 200 μL of 1.42 mM nitroblue tetrazolium (NBT). After the solution was preincubated at room temperature for 3 min, 100 μL of 0.5 U/mL xanthine oxidase was added to the mixture, and the volume was brought up to 3 mL with 50 mM phosphate buffer (pH 7.4). After the solution was incubated at room temperature for 20 min, absorbance was measured at 560 nm. The reaction mixture without xanthine oxidase was used as a blank (A1). The samples (A2) were added to the reaction mixture, in which O2^●− was scavenged and thereby inhibited the reduction of NBT. The absorbance was measured, and a decrease in O2^●− was represented by A2-A1. The scavenging activity of the superoxide anion radical (SRSA) was calculated using the following equation: SRSA %=(A2−A1/A1)×100. The scavenging activity of the samples (100 μg/mL) in DMSO on the hydroxyl radical (OH^●) was measured using the deoxyribose method [26] with slight modifications. The deoxyribose assay was performed in 10 mM phosphate buffer (pH 7.4) containing 2.5 mM deoxyribose, 1.5 mM H₂O₂, 100 μM FeCl₃, 104 μM EDTA, and a test sample (0.5 mg/mL). The reaction was initiated by adding ascorbic acid to a final concentration of 100 μM. The reaction mixture was incubated for 1 h at 37 °C in a water bath. After incubation, the color was developed by the addition of 0.5 % thiobarbituric acid followed by an addition of ice-cold 2.8 % trichloroacetic acid in 25 mM NaOH and incubation for 30 min at 80 °C. A control experiment was performed without the samples (A1). The samples (A2) were cooled on ice, and the absorbance was measured at 532 nm. The hydroxyl radical scavenging activity (HRSA) was calculated using the following equation: HRSA%=(A1− A2/A1)×100. The measurements were performed four times.

Cell culture

Murine 3 T3-L1 fibroblasts as preadipocytes were maintained in Dulbecco’s modified Eagle’s high-glucose medium (DMEM) containing 10 % calf serum, 100 units/mL penicillin, and 100 μg/mL streptomycin. Two-day post-confluent 3 T3-L1 cells, designated as day 0, were differentiated with complete DMEM containing 10 % FBS by adding a mixture (DMI) of 0.5 mM 3-isobutyl-1-methylxanthine, 100 μM indomethacin, 0.25 μM dexamethasone (DEX), and 167 nM insulin. The 3-isobutyl-1-methylxanthine, DEX, indomethacin, and Oil red O were purchased from Sigma-Aldrich (St. Louis, MO, USA). The medium was changed every 2 days. RCBs were added to the culture medium of the adipocytes on day 0. The cells were treated with 0, 50, or 150 μg/mL RCB extracts every day. After treatment with RCB on day 4 and day 7, the 3 T3-L1 adipocytes were harvested and lysed for Western blot analysis as described in section 2.12. To analyze cell viability, the cytotoxicity of the RCB was evaluated using 3-(4, 5-demethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) according to the manufacturer’s instructions.

Oil red O staining and triglyceride assay

Lipid accumulated within cells was visualized by Oil red O staining as described previously [27]. Oil red O staining was performed on day 7 of differentiation to stain accumulated lipid droplets in 3 T3-L1 adipocytes. The cells were gently washed with phosphate-buffered saline (PBS) and stained with filtered Oil red O solution (60 % isopropanol and 40 % water) for 30 min. After staining the lipid droplets red, the Oil Red O staining solution was removed, and the plates were rinsed with water and dried. The photographs were taken using an Olympus microscope (Tokyo, Japan). To analyze the content of cellular triglycerides, the cells were washed with PBS, scraped into 200 μL of PBS and sonicated for 1 min. The 3 T3-L1 cells were incubated with or without the selective PI3K inhibitor LY294002, which was purchased from Sigma-Aldrich (St. Louis, MO, USA) at a concentration of 10 μM in the presence or absence of RCB for 7 days. Total triglyceride content was measured in cell lysates using an assay kit, and cellular protein was measured using a Bio-Rad protein assay kit (Hercules, CA, USA). The results are expressed as percentage changes. All the experiments were carried out in triplicate.

RT-PCR

Total RNA was isolated from the 3 T3-L1 adipocytes using Trizol reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol. The total RNA (1 μg) was reverse-transcribed to cDNA using a reverse transcription system (Invitrogen, CA, USA) according to the manufacturer’s protocol. The mRNA expression of adipocyte differentiation-related genes was examined using cDNA and the following gene-specific primers: C/EBPβ, 5′-GACTACGCAACACACGTGTAACT-3′ and 5′-CAAAACCAAAAACATCAACAACCC-3′; PPARγ, 5′-TTTTCAAGGGTGCCAGTTTC-3′ and 5′-AATCCTTGGCCCTCTGAGAT-3′; C/EBPα, 5′-TTACAACAGGCCAGGTTTCC-3′ and 5′-GGCTGGCGACATACAGATCA-3′; β-actin (control), 5′-GACAACGGCTCCGGCATGTGCAAAG-3′ and 5′-TTCACGGTTGGCCTTAGGGTTCAG-3′.

Western blot analysis

Western blotting was performed according to standard procedures [6] with slight modifications. Briefly, whole cells were lysed in lysis buffer containing 50 mM Tris–HCl (pH 8.0), 0.4 % Nonidet P-40, 120 mM NaCl, 1.5 mM MgCl₂, 0.1 % SDS, 2 mM phenylmethylsulfonyl fluoride, 80 μg/ml leupeptin, 3 mM NaF and 1 mM DTT. Cell lysates were separated by 10 % SDS-polyacrylamide gel electrophoresis, transferred onto a polyvinylidene fluoride membrane (Amersham Pharmacia, England, UK), blocked with 5 % skim milk and hybridized with primary antibodies. The following antibodies were purchased from Cell Signaling technology (Danvers, MA USA): PPARγ, C/EBPβ, C/EBPα, aP2, FAS, Akt, and GSK3β; the monoclonal β-actin antibody was purchased from Chemicon (Millipore Inc., Billerica, MA USA). HRP-labeled mouse anti-rabbit IgG was obtained from Jackson ImmunoResearch. The Chemiluminescence kit was from Pierce (Rockford, IL). After incubation with horseradish-peroxidase-conjugated secondary antibody at room temperature, immunoreactive proteins were detected using a chemiluminescent ECL assay kit (Amersham Pharmacia, UK) according to the manufacturer’s instructions.

Measurement of glucose uptake

Glucose uptake in the 3 T3-L1 cells was measured using the fluorescent D-glucose analogue 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) (Invitrogen, Carlsbad, USA) with slight modifications to a previously reported protocol [28]. Briefly, 3 T3-L1 pre-adipocytes were differentiated in various concentrations (0, 50, and 150 μg/ml) of RCB or DMSO for 7 days in 6-well plates. After washing, the differentiated 3 T3-L1 cells were treated with or without the indicated concentrations of RCB and insulin in the absence or presence of 10 μM 2-NBDG. After incubation for 2 h, the cells were washed three times with PBS, and the resulting fluorescence was measured (excitation at 485 nm and emission at 530 nm) using a fluorescent microplate reader (Molecular Devices, USA). All the experiments were carried out in triplicate.

Animal experiments

Five-week-old Sprague–Dawley male rats were obtained from Central Lab Animal Inc. (Seoul, Korea). All animal experiments were conducted in accordance with the National Institutes of Health (NIH) guidelines and with the approval of the Animal Care and Use committee of Gyeongsang National University (Approval Number: GNU-140513-R0049). The rats were housed in polycarbonate cages in a room maintained at 22 °C with 55 % relative humidity on a 12 h dark/light cycle. All of the rats were allowed free access to food and water for five weeks. The rats were randomly divided into three groups that were fed a normal diet (ND, n=10), a high-fat diet (HFD, n=10), or a HFD supplemented with RCB (HFD+RCB, n=10) for 5 weeks. The ND group was maintained on a normal diet based on a commercial diet (#55VXT0038, Samyang Co, Korea). The HFD group was fed an HFD based on a commercial diet (rodent diet with 60 % kcal fat, Research Diet, Korea). To test the anti-obesity activity of RCB extracts (200 mg/kg BW) orally administered to the HFD-fed rats for five weeks. Food intake was measured daily, and body weight was measured every two days. Rats were food deprived overnight prior to blood collection and euthanized by ketamine (40 mg/kg) plus xylazine (2 mg/kg).

Group 1: Normal diet group (ND)

Group 2: High-fat diet group (HFD)

Group 3: HFD+RCB 200 mg/kg BW (HFD+RCB)

Biochemical and histological analysis

Body weight and fatty tissue mass (n=10 rats/group) were measured with sensitivity limits of 0.1 g and 0.01 g, respectively. Blood samples were collected and centrifuged 1000 x g for 15 min at 4 °C, and serum was separated to analyze plasma biomarkers. Epididymal fat pads were excised, weighed and stored at −20 °C until they were assayed. Concentrations of plasma triglyceride (TG), total cholesterol (TC), and high-density lipoprotein-cholesterol (HDL-C) were assayed enzymatically using commercial kits (Asan phams, Co., Korea) as previously described [27]. There were 10 animals per group in all experiments. Epididymal adipose tissue samples were dehydrated, embedded in paraffin, cut into 5-μm sections and stained with hematoxylin-eosin for microscopic assessment (Olympus, Tokyo, Japan). For quantification analysis, adipocyte dimensions were measured using MetaMorph image analysis software (Molecular devices, CA, USA) equipped for the automation and programmable distance and color segmentation (n=5 rats/group).

Inhibitory effect of RCB on 3 T3-L1 pre-adipocyte differentiation

To examine the effects of RCB on the differentiation of pre-adipocytes into adipocytes, confluent 3 T3-L1 pre-adipocytes were treated with various concentrations (0, 50, and 150 μg/ml) of RCB extract in the presence or absence of a DMI mixture. The cell culture media was supplemented with RCB extract from day 0 to day 7, and the fully differentiated cells exhibited numerous lipid droplets, indicating lipid accumulation. On the seventh day of incubation, lipid accumulation was examined as a marker of differentiation by Oil red O staining. RCB extract showed anti-adipogenic properties, as indicated by the decreased levels of Oil red O staining on differentiation day 7 in Fig. 1a. To further investigate whether RCB had an effect on adipocyte differentiation, lipid accumulation was quantified by measuring triglyceride content during 3 T3-L1 differentiation. The triglyceride content of the cells increased during 3 T3-L1 differentiation over the course of 7 days, whereas the addition of RCB extract into the differentiation media strongly blocked triglyceride accumulation (Fig. 1b). These results showed that treatment with 50 or 150 μg/ml RCB extract resulted in a 17 % or 28 % decrease, respectively, in triglyceride accumulation in the 3 T3-L1 adipocytes.

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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

Cytotoxic effects of RCB on 3 T3-L1 cells

The toxic concentration of the RCB extract was assessed via an MTT viability assay using thiazoyl blue tetrazolium bromide. 3 T3-L1 cells were treated with different concentrations of RCB (0, 50, and 150 μg/ml) in combination with a DMI mixture for either 4 or 7 days. There were no significant differences in 3 T3-L1 cell viability at concentrations of up to 150 μg/ml of RCB extract compared to the control (Fig. 1c). These results demonstrated that RCB strongly blocks adipocyte differentiation in 3 T3-L1 cells.

Inhibitory effect of RCB on adipogenic-specific gene and lipogenic gene expression

Adipocyte differentiation is accompanied by changes in the expression of various adipogenesis- and lipogenesis-related genes. C/EBPs and PPARγ are known adipogenic genes that have roles in transforming pre-adipocytes into mature adipocytes [5]. To investigate the anti-adipogenic mechanism of RCB, its effects on both mRNA and protein levels of C/EBPα, C/EBPβ, and PPARγ were elucidated. After inducing differentiation, 3 T3-L1 cells were exposed for 4 or 7 days to a concentration of 50 and 150 μg/ml RCB. As shown in Fig. 2a, the mRNA level of C/EBPβ was significantly lower in cells treated with 150 μg/ml RCB extract during adipocyte differentiation than that in the cells treated with DMI alone. The expression of C/EBPα and PPARγ mRNA was also significantly reduced following treatment with RCB extract (Fig. 2a). Furthermore, the protein levels of C/EBPβ, C/EBPα, and PPARγ were reduced in a dose-dependent manner after RCB treatment for 4 or 7 days (Fig. 2b). The activation of C/EBPα and PPARγ induces the expression of lipogenesis-related genes, including aP2 and FAS [5]. Thus, we examined whether the above-detailed down-regulation was related to decreased C/EBPα and PPARγ target gene expression levels. Consistent with the above observations, RCB suppressed the expression levels of C/EBPα and PPARγ target genes, such as aP2 and FAS (Fig. 2b). We also found that RCB extract treatment resulted in a dose-dependent suppression of aP2 and FAS at the protein level. Taken together, these results suggest that the activation of C/EBPα and PPARγ plays a critical role in the regulation of adipogenesis by RCB.

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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

Effect of RCB on the phosphorylation of Akt and GSK3β during adipocyte differentiation

To investigate whether Akt, a major factor in adipocyte differentiation, is regulated by RCB during 3 T3-L1 differentiation, levels of phosphorylated Akt and GSK3β were analyzed and compared with total levels of Akt. Our results showed that DMI-stimulated 3 T3-L1 adipocytes exhibited strongly increased phosphorylation levels of Akt (Ser473) and GSK3β (Ser9). The phosphorylation of Akt was significantly decreased in the RCB-treated groups compared to the differentiated 3 T3-L1 adipocytes (Fig. 3a). Likewise, the expression of phospho-GSK3β was also substantially decreased following treatment with RCB during 3 T3-L1 cell differentiation (Fig. 3b). These results suggest that RCB extract treatment decreases Akt phosphorylation and down-regulates the phosphorylation of GSK3β, a substrate kinase of Akt, leading to the inhibition of the adipogenesis pathway.

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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

RCB inhibited 3 T3-L1 adipocyte differentiation through the Akt signaling pathway

To further understand the underlying mechanism of the inhibitory effects of RCB on adipocyte differentiation, we performed an experiment with LY294002, a chemical inhibitor of the Akt pathway. After treatment with DMI for 7 days, differentiated 3 T3-L1 cells showed increased accumulation of triglyceride droplets compared to undifferentiated 3 T3-L1 cells (Fig. 3c). Interestingly, treatment with 10 μM LY294002 resulted in a decrease in intracellular triglyceride accumulation in the differentiated 3 T3-L1 cells. Moreover, we found that the inhibitory effects of RCB on the formation of lipid droplets were more enhanced by the combined treatment of LY29400 and RCB than by treatment with RCB alone (Fig. 3c). These results indicate that the role of Akt signaling is significantly associated with the functional modulation of the RCB, which reduces adipogenesis in 3 T3-L1 cells.

RCB reduced glucose uptake in 3 T3-L1 adipocytes

To examine the effects of RCB on glucose uptake in 3 T3-L1 adipocytes, a fluorescent deoxyglucose analog (2-NBDG) was used to calculate glucose uptake rates. 3 T3-L1 cells were exposed to 0, 10, 50, and 150 μg/ml concentrations of RCB extract in differentiation media for 7 days. The RCB treatment led to a significant dose-dependent decrease in glucose uptake compared to cells treated with DMI alone; glucose uptake in the 50 and 150 μg/ml RCB-treated groups showed decreases of 27 % and 38 %, respectively, compared to the DMI alone group (Fig. 3d).

RCB induced decreases in body weight and adipose tissue weight in HFD-induced obese rats

We further evaluated whether RCB possessed anti-obesity effects in rats fed a HFD for 5 weeks. At the end of the experiment, the body weights of the HFD group were 39 % greater than those of the ND control group, demonstrating that HFD-fed rats developed obesity (Additional file 1 and Fig. 4a). The weight gain observed for the group of rats that were administered RCB extract (200 mg/kg/daily) was markedly decreased compared to the HFD only group. We did not observe any significant changes in food intake in any of the treatment groups compared to the HFD-fed group and the HFD-fed plus RCB extract treatment group (data not shown). Thus, these data indicate that the reduction in body weight gain in the RCB-treated rats was not mediated by a reduction of food and water intake in the HFD rats. To investigate whether the reduction in body weight gain in the RCB-treated group was related to decreased fat accumulation, epididymal and perirenal adipose tissues were weighed. Supplementation with RCB significantly reduced epididymal fat (25 %) and perirenal fat (22 %) mass compared to the untreated HFD group (Fig. 4b and c). Histological examination of the epididymal adipose tissue revealed that adipocyte size was significantly smaller in the epididymal adipose tissue of the RCB-treated group than in that of the HFD only group (Fig. 4d), further demonstrating that the reduction in body weight gain primarily arose because of decreased fat accumulation and adipocyte size.

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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

This work was supported by the Ministry of Education, Basic Science Research Program, National Research Foundation, Korea (No. 2014045966).