Black Raspberry (Rubus coreanus Miquel) Promotes Browning of Preadipocytes and Inguinal White Adipose Tissue in Cold-Induced Mice

Dulbecco modified eagle medium (DMEM) medium and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, NY, USA). Insulin, 3-isobutylmethylxanthine (IBMX), indomethacin, 3,3′,5-triiodo-L-thyronine (T3), dexamethasone, troglitazone, Oil-red O powder, and CL316,243 were obtained from Sigma Aldrich (St Louis, MO, USA). Poly-vinylidene difluoride (PVDF) was procured from Millipore (Merck KGaA, Darmstadt, Germany). The electrochemiluminescence (ECL) kit was obtained from GE Healthcare Life Sciences (Seoul, Korea).

Dried BR was provided by Kyung Hee University Korean Hospital (Seoul, Korea). BR water extract (BRWE) was obtained by extracting BR in hot water at 100 °C for 3 h, followed by filtering (No 4, Whatman, Kent, UK). After being freeze-dried in a vacuum, it was dissolved in DMSO (20 mg·mL⁻¹).

The human mesenchymal stem cells (hMSCs) were purchased from the Cell Engineering for Origin (Seoul, Korea). The cells were cultured in DMEM media supplemented with 10% FBS and 100 U mL⁻¹ of penicillin and streptomycin in a CO₂ incubator at 37 °C with 5% CO₂ until confluence. The cells, on two days after confluence, were differentiated with differentiation medium (0.5 mM IBMX, 1 µM dexamethasone, 1 µg·mL⁻¹ insulin, and 100 nM indomethacin) that was added to DMEM containing FBS 10% for two days (Days 2). Then, the medium was replaced by maintenance medium containing 1 µg·mL⁻¹ insulin, 50 nM T3, and 0.5 µM troglitazone once every two days (three times, Days 2–6). On Day 2, BRWE was prepared in a maintenance medium at concentration of 5 and 10 µg mL⁻¹.

3T3-L1 cells, a mouse embryo fibroblast cell line, were obtained from the American Type Culture Collection (Rockville, MD, USA). The cells were cultured in DMEM media supplemented with 10% FBS and 100 U mL⁻¹ of penicillin and streptomycin in a CO₂ incubator at 37 °C with 5% CO₂ until confluence. The cells, two days after confluence, were differentiated with differentiation medium (0.5 mM IBMX, 0.5 µM dexamethasone, 1 µg·mL⁻¹ insulin, 50 nM T3, and 0.5 µM troglitazone) and were added to DMEM containing FBS 10% for two days (Days 2). Then, the medium was replaced by maintenance medium containing 1 µg·mL−1 insulin, 50 nM T3, and 0.5 µM troglitazone once every two days (three times, Days 2–6). On Day 2, BRWE was prepared in a maintenance medium at concentration of 5 and 10 µg·mL⁻¹. For full differentiation of white adipocytes, 3T3-L1 cells were cultured and differentiated without troglitazone.

The cell cytotoxicity was measured with a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) kit (Promega, Madison, WI, USA) as previously described [13].

Intracellular lipid accumulation was measured using Oil Red-O, as described previously [12].

RNA isolation and real-time RT-PCR were performed as previously described [14]. Briefly, the total RNA was obtained using GeneAll^R RiboEX Total RNA extraction (GeneAll Biotechnology, Seoul, Korea). The relative gene expressions were calculated based on the comparative CT method using the StepOne software v2.1 (Applied Biosystems, Foster City, CA, USA). The mRNA expression of Gapdh was used as an endogenous control. The primers used in this study are as follows: Ucp1 (F: 5′-AACTGTACAGCGGTCTGCCT-3′, R: 5′-TAAGCCGGCTGAGATCTTGT-3′), Pgc1a (F: 5′-AATGCAGCGGTCTTAGCACT-3′, R: 5′-TGTTGACAAATGCTCTTCGC-3′), and Gapdh (F: 5′-AACTTTGGCATTGTGGAAGG-3′, R: 5′-GGATGCAGGGATGATGTTCT-3′).

Male C57BL/6J mice (7-week-old) were purchased from Deahan Biolink Co. (Eumsung, Korea) and kept for 1 week prior to the experiments. The mice were orally administrated BRWE (100 mg·kg⁻¹, daily) or an equivalent volume of vehicle (5% DMSO in phosphate buffered-saline (PBS)) for 2 weeks. The mice were kept at 4 °C, and their rectal temperature was measured at the indicated time points. The mice were sacrificed by cervical dislocation under CO₂ asphyxiation. The animal experiments regarding mice were performed according to a protocol approved by the Animal Care and Use Committee of the Institutional Review Board of Kyung Hee University (confirmation number: KHUASP (SE)-13-012).

Zebrafish were raised according to standard protocol [15]. Larvae obtained from the adult zebrafish crosses were raised in a 28.5 °C incubator and fed regular diets starting at 5.5 days post fertilization (dpf). BRWE at 100 ug·mL⁻¹ was treated and replaced daily in fish water for 8 days starting from 10 dpf. For adipocyte visualization, the larvae were incubated for 30 min in dark in Nile red (Invitrogen) prepared from a stock solution (1.25 mg·mL⁻¹ in acetone) by 1:2500 dilution. After rinsing with fish water several times, the larvae were viewed using a Leica M165FC microscope equipped with Leica DFC500. The captured fluorescent signals of Nile red were quantified using Image J 1.51v. Student’s t-test was used to determine statistical significance (p < 0.05). The experimental protocols used in the zebrafish study were approved by the committee for Ethics in Animal Experiments of Wonkwang University (WKU15-151).

WAT was washed in PBS and fixed in 10% formalin for 2 weeks. Then, the tissues were embedded in paraffin. The tissue sections were deparaffinized in xylene and rehydrated with ethanol/water and then stained with hematoxylin-eosin (H&E). Microscopic examinations were performed, and photographs were taken under a regular light microscope. The average adipose droplet size was calculated using the Image J software program (National Institute of Health, Bethesda, MD, USA).

The cells and tissues were lysed through radioimmunoprecipitation assay (RIPA) buffer (Cell Signaling Technology, Danvers, MA, USA) on ice for 30 min, and then, insoluble materials were removed by centrifugation at 13,000 rpm for 20 min at 4 °C. The lysates were resolved by sodium dodecyl sulfate (SDS)-polyacrylamaide gel electrophoresis and transferred onto a PVDF membrane. Then, the membranes were blocked in 5% skim milk and incubated with the respective primary antibody (1:1000) overnight at 4 °C followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000) for 1 h at room temperature. The protein signals were detected using the ECL advance kit.

The cells and tissues were fixed using 10% formalin and blocked with 5% BSA for 1 h. After, the cells and tissue were incubated with the indicated primary antibodies (anti-UCP1 and anti-PGC1α, 1:50 in 5% BSA) overnight at 4 °C. After washing, the cells and tissue were incubated with Alexa Flour 488- or 633-conjugated secondary antibody (1:1000), and the fluorescence was detected using an EVOS^R Cell Imaging systems (Thermo Scientific, Carlsbad, CA, USA).

Data were expressed as mean ± SEM of independent experiments. Statistical differences were calculated by one-way ANOVA and a subsequent post hoc Tukey test unless stated otherwise. All statistical analyses were completed using SPSS statistical analysis software version 11.5 (SPPS Inc., Chicago, IL, USA). All probability values (^#p < 0.05, * p < 0.05, ** p < 0.01, and *** p < 0.001,) were used as the criterion for statistical significance.

Cytotoxicity of BRWE in hMSCs was confirmed in our previous report [11]; thus, we chose the concentrations of 5 and 10 μg·mL⁻¹ for the treatment of the pre-adipocytes. To determine the anti-adipogenic effect of BRWE, we assessed the intracellular lipid accumulation during the differentiation of pre-adipocytes to adipocytes in hMSCs cells. As a result, BRWE attenuated the development of lipid droplets in a dose-dependent manner during white adipocyte differentiation (Figure 1A,B). Next, it was confirmed whether BRWE treatment potentially affects the expression of adipogenesis-related proteins such as peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα). As shown in Figure 1C, BRWE treatment suppressed the mRNA levels of Pparg and Cebpa in 3T3-L1 adipocytes. Furthermore, by immunoblotting assays, we observed a noticeable decrease of the levels of PPARγ and C/EBPα reduction in BRWE-treated white adipocytes (Figure 1D,E).

Furthermore, to investigate the role of BRWE during adipocyte development, we treated 100 μg·ml^-1 BRWE or 0.1% DMSO to zebrafish larva for 8 days from 10 to 17 days dpf. Zebrafish is a widely accepted experimental model for adipogenesis study because they develop rapidly, they have a short life cycle, and the genome sequence information is in detail enough for pathway investigations [16] and because lipid metabolism pathways are conserved between mammals and fish [17,18,19]. To analyze the amount of lipid, the larvae were stained with Nile red and photographed. In addition, the length of each larva was measured, since adipocyte development is known to be proportional to the size of larvae [20]. The average length of larvae did not differ significantly between the BRWE-treated group and the vehicle-treated group (5.98 and 6.03 mm in the BRWE-treated or control groups, respectively), and the range of larvae was from 5.5 to 6.5 mm. However, signal intensity of lipid visualized by Nile red displayed more than 8 times reduction in the BRWE-fed group when compared to the control group (Figure 1C,D). This result indicated a repressive effect of BRWE on white adipocyte development in zebrafish. Taken together, the treatment of BRWE had a significant suppressive impact on lipid accumulation during adipocyte development.

Cold stimulation is widely known to affect remodeling of adipose tissues, i.e., BAT activation and WAT browning [21]. To confirm the effect of BRWE on the capacity of cold tolerance, we treated C57BL/6J with BRWE (100 mg·kg⁻¹) by oral gavage for 12 days and conducted a cold stress test at 4 °C for 5 h. After 5 h of acute cold exposure, the rectal temperature of PBS-fed mice declined from 32.77 ± 0.41 °C and their body temperature returned to a normal state (36–37 °C) at room temperature (RT, 25 °C) after 10 min. In the BRWE-fed mice, temperature decline was significantly improved as it maintained 34.4 ± 0.54 °C of rectal temperature after 5 h, and the recuperative power of body temperature also improved compared to the PBS-fed mice (Figure 2A–C). However, body weight change and adipose tissue weight in mice with or without BRWE treatment showed no significant difference (Figure 2D–F). The results also showed that BRWE treatment affected iWAT browning. The H&E staining and bar graph shown in Figure 2G,H shows the reduced lipid droplet size in iWAT. Immunofluorescence staining was performed in iWAT of BRWE-fed or PBS-fed mice, and we could verify an extensively strong signaling of the UCP1 (green dye) displayed in BRWE-treated mice compared to the PBS-fed mice (Figure 2I,J). The beige-specific markers such as PR domain zinc-finger protein 16 (PRDM16), PGC1α, UCP1, and TBX1 were increased in iWAT of BRWE-fed and cold-exposed mice (Figure 2K,L). Furthermore, we observed the thermogenic alteration of BRWE in other adipose tissues (epididymal WAT and BAT). BRWE activated the UCP1-related thermogenic signaling in BAT while it had no effect on UCP1 expression in epididymal WAT of cold-exposed mice (Supplementary Figure S1A,B).

To study the efficacy of BRWE on beige induction in 3T3-L1 murine pre-adipocytes, we attempted to measure the intracellular lipid accumulation in DM (Be)-stimulated 3T3-L1 cells. The treatment with BRWE (5 and 10 μg·mL⁻¹) did not affect accumulation of intracellular lipid in DM (Be)-stimulated 3T3-L1 cells (Figure 3A,B); however, we found out that mRNA expressions of thermogenic factors such as Ucp1 and Pgc1a were markedly upregulated by BRWE (Figure 3C–E). In accordance with the real-time RT-PCR results, we further confirmed that the BRWE treatment also increased the expression of beige-adipocyte related proteins by using western blot and immunofluorescence staining. BRWE treatment increased the protein expression of beige markers (UCP1 and PGC1α) compared to the DM (Be) only control group (Figure 3F).

Browning within WAT can occur due to new preadipocytes that differentiate into beige adipocytes or result from trans-differentiation of mature white adipocytes into beige adipocytes [22]. Above, we could conclude BRWE can induce preadipocytes to differentiate into beige adipocytes. Thus, we next attempted to find out whether BRWE has the capability to induce browning of mature white adipocytes. Accordingly, BRWE treatment with 48 h increased the UCP1 levels in mature 3T3-L1 adipocytes of which the level was similar to the effect of the β3 AR agonist CL316,243 (Supplementary Figure S2).

Although non-shivering thermogenesis is mediated by UCP1, activation of UCP1 may not necessarily mean equal to the induction of mitochondrial thermogenesis program [23]. Thus, we measured further factors related in mitochondrial biogenesis and activation. As shown in Figure 3G, a Mitotracker staining assay revealed increased abundancy of mitochondria in BRWE-treated cells. Increased factors of mitochondrial abundance and action including cell death activator (CIDEA) [24] and nuclear respiratory factor 1 (NRF1) [25] showed the increased mitochondrial biogenesis/activation induced by BRWE, and furthermore, the increased mitochondria enzyme carnitine palmitoyltransferase 1B (CPT1B) [26] indicated increased mitochondrial β-oxidation by BRWE treatment (Figure 3H).

In our previous study, we confirmed the phytochemical profile of BRWE using HPLC MS/MS analysis which identified four major compounds (4-hydroxybenzoic acid (4-HA), ellagic acid (EA), gallic acid (GA), and salicylic acid (SA)) [12]. Treatment of aforementioned compounds in vitro were carried out to evaluate their potential effects on the expression of beige-specific markers. First, the cytotoxicity of these constituents was measured. Murine 3T3-L1 pre-adipocytes were treated with various concentrations (12.5 to 100 µM) of the four major compounds for 48 h. As shown in Figure 4A, the cell viability was not affected up to the concentration of 100 µM of all compounds. We next investigated whether four compounds treatment could affect beige markers (PGC1α, UCP1, TBX1, and NRF1) in DM (Be)-stimulated 3T3-L1 cells. Interestingly, the results showed that EA-treated 3T3-L1 cells significantly increased the beige markers when compared against DM (Be)-stimulated 3T3-L1 cells (Figure 4B,C), while the other three constituents (4-HA, GA, and SA) failed to display such an effect.