Artepillin C, a Typical Brazilian Propolis-Derived Component, Induces Brown-Like Adipocyte Formation in C3H10T1/2 Cells, Primary Inguinal White Adipose Tissue-Derived Adipocytes, and Mice

The purity of all chemicals was >98%. ArtC was isolated and purified from Brazilian propolis [16, 17]. In brief, propolis originating from Baccharis dracunculifolia was collected in Minas Gerais, Brazil in 2008, and stored at 4°C until use. The propolis sample was extracted with methanol, and pure ArtC was isolated from the extract by repeated column chromatography. The purity of the compound was >99% as determined from its ¹H-NMR spectrum. Dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), triiodothyronine (T3), and cycloheximide were obtained from Wako Pure Chemical Industries (Osaka, Japan). Rabbit polyclonal β-actin antibody (#4967) and rabbit polyclonal cytochrome c oxidase subunit IV (CoxIV) antibody (#4844) were obtained from Cell Signaling Technology (Beverly, MA). Rabbit polyclonal UCP1 antibody (ab10983) and rabbit polyclonal PRDM16 antibody (ab106410) were obtained from Abcam (Tokyo, Japan).

The murine C3H10T1/2 cell line (IFO050415, Health Science Research Resources Bank, Osaka, Japan) was a gift (April, 2013) from Dr. Hitoshi Yamashita, Chubu University, Japan. This cell line is a stem cell that can be differentiated to brown-like adipocytes [20]. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere with 5% CO₂. Confluent cells were placed in a differentiation medium (DMEM containing 10% FBS, 1 μM dexamethasone, 0.5 mM IBMX, 3 nM T3, and 2 μM insulin) with or without various compounds for 2 days before the medium was changed to induction medium (DMEM containing 10% FBS, 3 nM T3, and 2 μM insulin) with or without various compounds. The medium was changed every 2 days and the cells were cultured for an additional 6 days to obtain mature adipocytes.

Male C57BL/6J mice (7–9 weeks of age) were obtained from Japan SLC (Shizuoka, Japan) and housed in an animal room (23 ± 3°C, 12 h light:dark cycle, lights on from 8:00 to 20:00 h) and allowed ad libitum access to water and a standard laboratory diet (CE-2, CLEA Japan, Tokyo, Japan) [21]. After 1 week of breeding, mice were killed and iWAT was carefully removed and the SVF was isolated [22]. Briefly, isolated iWAT was minced and suspended and digested in 0.2% collagenase (125 collagen digestion units/mL, C6885, Sigma-Aldrich Japan, Tokyo, Japan) solution at 37°C for 30 min. After digestion, the cell suspension was filtered using a cell strainer and centrifuged. The obtained pellet (SVF) was re-suspended in DMEM supplemented with 10% FBS, and plated on collagen-coated dishes (CORNING, NY). Confluent cells were differentiated and induced to adipocytes as well as C3H10T1/2 cells. The experimental design was approved by the Animal Experiment Committee, Chubu University, and the mice were maintained in accordance with their guidelines (Permission No. 2610031 and 2710011).

At 8 days (C3H10T1/2 cells) or 12 days (primary adipocyte) after the differentiation and induction of preadipocytes to adipocytes, total adipocyte RNA was isolated using QIAzol TM reagent (QIAGEN, Tokyo, Japan) according to the manufacturer’s directions. As in our previous studies [23, 24], total RNA (1.0 μg) was reverse transcribed to cDNA and gene expression was quantified with a real-time PCR system (ABI PRISM 7300 Sequence Detection System; Life Technologies, Tokyo, Japan). The assay identification numbers of the TaqMan gene expression assays were: UCP1, Mm01244861_m1; TATA box binding protein (TBP), Mm00446971_m1; cell death-inducing DNA fragmentation factor, alpha subunit-like effector A (Cidea), Mm00432554_m1; cytochrome C oxidase subunit VIII b (Cox8b), Mm00432648_m1; elongation of very long chain fatty acids-like 3 (Elovl3), Mm00468164_m1; and β3-AR, Mm02601819_g1. The gene expression level was expressed relative to the control (= 1.0) after normalization using the TBP gene expression level.

After various treatments or incubations, the cells were lysed [25]. Aliquots of the supernatant were treated with Laemmli sample buffer for 5 min at 100°C [26]. As in our previous study [21], samples (50 μg protein) were loaded into an SDS-PAGE system and resulting gels were transblotted onto PVDF membranes before the sheets were probed with various antibodies for 16 h at 4°C, and reacted with horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibody. Immunoreactivity was then visualized using Pierce Western Blotting Substrate (Thermo Fisher Scientific, Yokohama, Japan) [21]. All experiments were performed in triplicate, and representative results are shown.

C3H10T1/2 cells were differentiated into mature adipocytes in the presence or absence of ArtC as described above. At 8 days after differentiation, the cells were incubated in medium containing 20 μg/mL cycloheximide and harvested at different time points and under various conditions, as indicated [27]. After the indicated period, the cells were lysed and immunoblot analysis for PRDM16 protein was performed as described above.

C57BL/6J mice (male, 4 weeks of age) were obtained from Japan SLC. The mice were housed in an animal room (23 ± 3°C, 12 h light:dark cycle, lights on from 8:00 to 20:00 h) and allowed ad libitum access to water and a standard laboratory diet (CE-2) [21]. After 1 week of breeding, mice were divided into three groups: control (vehicle, 5% DMSO–4% Tween 80 in water), low-dose (5 mg/kg) and high-dose (10 mg/kg) ArtC. Vehicle or ArtC was orally administered to mice by direct stomach intubation every day for 4 weeks. Then, the mice were killed and blood samples were collected. iWAT, BAT, and epididymal WAT (eWAT) were removed and weighed. Small pieces of adipose tissues were then fixed and used for histological analysis. Aliquots of tissues were homogenized and the mitochondrial fraction was isolated using the method of Lau et al. [28], and immunoblot analysis was performed as described above. The experimental design was approved by the Animal Experiment Committee, Chubu University, and the mice were maintained in accordance with their guidelines (Permission No. 2710011).

Adipose tissues were fixed in 4% paraformaldehyde for 24 h at 4°C. Then the tissues were embedded in paraffin and cut into slices for UCP1 (5-μm thick) and PRDM16 (3-μm thick) immunostaining, and deparaffinized and rehydrated using xylene, ethanol, and water. Adipose tissue sections were stained with H&E (both from Sakura Finetek Japan, Tokyo, Japan) or UCP1 and PRDM16 immunostained. For immunohistochemistry, slides were submerged in 0.1 M sodium citrate (pH 6.0) and heated to 100°C for 15 min in a laboratory microwave (500 W). Slides were incubated with 0.3% hydrogen peroxide for 30 min to block endogenous peroxidase, and 2.5% normal horse serum (included in kit) for 20 min at room temperature. Slides were then incubated with anti-UCP1 antibody (1:500) and anti-PRDM16 antibody (1:150) for 16 h at 4°C, and with anti-rabbit IgG (included in kit) for 30 min at room temperature. Labeling was visualized with 3, 3′-diaminobenzidine (DAB) as the chromogen using ImmPress kit and Immpact DAB peroxidase substrate kit (Vector Laboratories, Burlingame, CA) according to the manufacturer’s directions. Then, sections were counterstained with hematoxylin.

Plasma NE concentration was measured using an ELISA specific for NE (ELISA kit, Cloud-Clone Corp., TX) according to the manufacturer’s instructions.

All data are expressed as the means ± SEM. Differences between means were compared by the Tukey-Kramer test or Student’s t-test. For all statistical tests, differences with P values < 0.05 were considered significant.

We first examined whether ArtC induced brown-like adipocytes using two cellular systems. C3H10T1/2 cells were differentiated into adipocytes in the presence or absence of ArtC, and the mRNA level of brown-like adipocytes markers was assayed. Administration of ArtC significantly increased the mRNA levels of UCP1, Cidea, Cox8b, and Elovl3 in a dose-dependent manner (Fig 2A). The use of primary adipocytes derived from the SVF of isolated iWAT is a suitable evaluation method for inducing the browning of white adipocytes in response to drugs or food-derived factors [22]; likewise, using C3H10T1/2 cells is a suitable method for inducing development of brown-like adipocytes. Administration of ArtC during the differentiation significantly induced the brown-like adipocytes implicated-gene expression levels in primary iWAT-derived adipocytes (Fig 2B).

Next, we confirmed that ArtC induces UCP1 and PRDM16 proteins, and functions as an inducer of brown-like adipocytes via its action as a PPARγ agonist. The administration of ArtC clearly induced UCP1 and PRDM16 proteins in a concentration-dependent manner (Fig 3A). Furthermore, the administration of T0070907 (a selective PPARγ antagonist) during differentiation resulted in inhibition of ArtC-induced UCP1 and PRDM16 protein expression (Fig 3B). Recent studies showed that rosiglitazone induced browning of the white adipocytes via not only activation of PPARγ but also stabilization and accumulation of the PRDM16 protein [27], and the mechanism of ArtC-induced brown-like adipocyte formation may be similar to that of rosiglitazone. Therefore, we examined the effect of PRDM16 protein stabilization on ArtC-induced brown-like adipocytes. C3H10T1/2 cells were differentiated with or without ArtC for 8 days, and then the cells were treated with cycloheximide and the degradation of PRDM16 protein was examined. ArtC clearly inhibited degradation of PRDM16 protein and induced stabilization (Fig 4A and 4B). In addition, ArtC extended the half-life of PRDM16 protein from 6.2 to 19.2 h (Fig 4C). Next, C3H10T1/2 cells were differentiated without ArtC for 8 days, and then the cells were treated with vehicle or ArtC. Treatment of the cells with ArtC also induced significant stabilization, accumulation of the PRDM16 protein compared to vehicle (Fig 4D and 4E), and extended the half-life of PRDM16 protein from 9.4 to 18.2 h (Fig 4F).

As expected, the results obtained from C3H10T1/2 cells and primary iWAT-derived adipocyte studies demonstrated that administration of ArtC induced brown-like adipocyte formation in mice. To confirm whether oral administration of ArtC induces brown-like adipocyte formation in mice, ArtC was administered to mice for 4 weeks, then UCP1 protein expressions in iWAT, eWAT, and BAT were examined using immunohistochemistry and immunoblotting. Body weight gain, food intake, and adipose tissues weight (iWAT, eWAT, and BAT) were not affected by the administration of ArtC (S1 Table). The H&E staining of iWAT obtained from the ArtC groups clearly showed multilocular adipocytes which imply that the morphological characteristic of brown-like adipocytes vary in a dose-dependent manner (Fig 5A). Moreover, these multiocular adipocytes showed distinct immunostaining of UCP1 and PRDM16 (Fig 5A). By contrast, multilocular adipocytes and UCP1-immunopositive cells were not observed in eWAT of the ArtC groups (Fig 5B). H&E staining and UCP1 and PRDM16 immunostaining did not differ in BAT from the three groups (Fig 5C). To further confirm the results obtained from immunohistochemistry and studies of mitochondrial function, the expression levels of UCP1 and CoxIV protein were examined in iWAT and BAT. The expression levels of UCP1 and CoxIV protein were significantly elevated in iWAT of mice administered 10 mg/kg ArtC compared to the control group (Fig 6A). However, the expression levels in BAT of both UCP1 and CoxIV did not differ among the three groups (Fig 6B). Further, the plasma NE concentration and β3-AR mRNA level in iWAT and BAT were not significantly affected by the administration of ArtC (Fig 7).