Novel discovery of Averrhoa bilimbi ethanolic leaf extract in the stimulation of brown fat differentiation program in combating diet-induced obesity

3 T3-L1 preadipocytes and C2C12 myoblasts were purchased from the American Type Culture Collection (Manassas, VA, USA). They were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% bovine serum and 10% fetal bovine serum (FBS), respectively. Resazurin-based assay was used to determine the cytotoxicity of the extract. Both cell types were treated with DBB for 3, 6, and 9 days at concentrations range from 6.25–200 μg/ml. Cell viability was assessed by the reduction of resazurin, a non-fluorescent, blue-coloured soluble dye to a highly fluorescent pink resorufin in response to the population of cell metabolism. Resorufin was measured at an excitation wavelength of 570 nm.

The differentiation of adipocytes was initiated 2 days after the cells reached confluence by adding 0.5 mM isobutyl methylxanthine (IBMX), 1 μM dexamethasone (DEX) and 1 μg/ml insulin to DMEM containing 10% FBS. 2 days later, the differentiation medium was replaced by DMEM supplemented with 10% FBS and 1 μg/ml insulin. The differentiated cells were stabilised in DMEM containing 10% FBS for another 2 days. C2C12 myoblasts were induced to differentiate by substituting the supplements of 10% FBS with 10% horse serum once the cells reached confluence. Fresh differentiation medium was replaced every 2 days until the formation of myotubes were completed. Both differentiation methods were performed according to the manufacturer’s instruction.

C2C12 myoblasts were cultured in DMEM supplemented with 10% FBS. Brown adipocyte differentiation was initiated after the cells reached confluence by adding 0.5 mM IBMX, 1 μM DEX, 1 μg/ml insulin and 1 μM rosiglitazone (ROSI). Two days later, the differentiation medium was replaced by DMEM supplemented with 10% FBS, 1 μg/ml insulin and 1 μM ROSI. These differentiated cells were stabilised in DMEM containing 10% FBS for another 2 days until oil droplets formation was observed. The brown adipocyte differentiation was done by adopting the method of Sharma et al. [9] with slight modifications.

To visualise the presence of adipocytes, cells were washed once with PBS and fixed with 4% paraformaldehyde for 10 min. Lipid droplets produced by adipocytes were stained by Nile Red solution. The trapped Nile Red solution between lipid droplets was visualised by the IN Cell 2200 Analyzer High Content Screening System (GE Healthcare, PA, USA) at an excitation wavelength of 460 nm.

3 T3-L1 preadipocytes were cultured in CellCarrier-96 black tissue culture plate (Perkin Elmer) and initiated adipocyte differentiation according to the standard protocol. After 7 days of differentiation, 3 T3-L1 adipocytes were treated with DMEM and 10% FBS containing either 100 μg/ml DBB, 1 μM ROSI or 0.5% DMSO. Cell medium containing all supplemented agents and treatment were refreshed every 2–3 days and maintained for 10 days. At day 10, the treated cells were washed with PBS and fixed with 4% paraformyldehyde. The cells were then blocked with 1% BSA before overnight incubated with primary antibody at 4 °C. Primary antibodies at 1:100 dilution used were Human/Mouse PRDM16 Sheep antibody (AF6295, R & D Systems), PGC1-alpha Goat antibody (GTX89046, GeneTex) UCP1/2/3 (FL-307) rabbit antibody (sc 28,766, Santa Cruz) and Anti-FNDC5 rabbit antibody (AB131390, Abcam). Secondary antibody incubation was conducted on the following day. The treated cells were incubated with host specific secondary antibody conjugated with fluorescent dye at 1:200 dilutions for 1 h at 37 °C. Secondary antibodies used were Thermo Fisher Pierce Rb anti-sheep IgG (H + L), FITC conjugated, Thermo Scientific Pierce Rb anti-goat IgG (H + L) secondary antibody, FITC conjugate and Santa Cruz Goat anti-rabbit IgG-CFL 488. The full plate containing treated cells was then subjected to imaging using the IN Cell Analyzer High Content Screening System. The fluorescent intensity produced from the captured cell images was then quantified using the IN Cell Developer software. Data was presented as fold increase of fluorescent intensity with relative to 0.5% DMSO treated cells.

C2C12 myoblasts were cultured until reaching 70% confluence. Fresh culture medium was then substituted with supplemented DMEM containing serially diluted DBB extracts ranging from 50 to 200 μg/ml. Vehicle-treated samples contained 0.5% DMSO without DBB. Cells were maintained for 7 days with fresh treated medium replenished every 2–3 days. Differentiated 3 T3-L1 adipocytes were treated with DMEM and 10% FBS containing serially diluted DBB extracts ranging from 50 to 200 μg/ml. Cells were maintained for 7 days with fresh treated medium replenished every 2–3 days. Vehicle-treated samples contained 0.5% DMSO without DBB. Cellular proteins were extracted by using M-PER® Mammalian Protein Extraction Reagent (Thermo Scientific Scientific, Waltham, MA). Protein concentrations of the lysed samples were determined by the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). 20 μg aliquots of protein were subjected to the sodium dodecyl sulfate polyacrylamide gel electrophoresis separation. The chromatographically separated proteins were transferred to a polyvinylidine difluoride membrane using the iBlot™ Gel Transfer Device (Thermo Fisher Scientific). After the transfer, the membrane was immersed in a blocking solution containing Phosphate Buffer Saline with 0.1% Tween-20 (PBS/T) and 3% BSA for 1 hour, followed by overnight primary antibody incubation. Primary antibodies at 1:1000 dilution used were PRDM 16 Polyclonal Antibody (PA5–20872, Thermo Fisher Scientific) and β-Actin (13E5) Rabbit mAb (#4970, Cell Signaling Technology). Then, the membrane was washed 3 times with PBS/T, 5 min each time before incubated with secondary antibody in the blocking solution at room temperature for 1 hour. Secondary antibody at dilution 1:2000 used was Goat Anti-Rabbit IgG-HRP (sc 2030, Santa Cruz). The signal of bound antibody was then enhanced by membrane immersion into the Clarity™ ECL Western Blotting Substrate (Bio-Rad, Hercules, CA) for 5–10 min. The bound antibody was then detected using the ChemiDoc XRS+ imaging system (Bio-Rad) and qualified with Image Lab 5.2.1 (Bio-Rad) image analysis software.

The Mito Stress Test assay was conducted to determine the basal metabolic rate (BMR), mitochondrial respiratory capacity (MRC), as well as reserve respiratory capacity (RRC) of adipocytes upon DBB treatments. 3 T3-L1 preadipocytes were cultured and differentiated in the culture plate provided by the Seahorse XF Cell Mito Stress Test Kit (Agilent Technologies, Santa Clara, CA). The differentiated cells were treated with 100 μg/ml DBB for 7 days. On day 8, the Mito Stress Test assay was carried according to the manufacturer’s instructions. The Seahorse XFe24 Analyzer (Agilent Technologies, Santa Clara, CA) was used to measure mitochondrial function in cells. This assay contained four pre-weighted mitochondrial drugs: oligomycin (oligo), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrozone (FCCP), rotenone and antimycin A (rot/AA). The machine measured the baseline cellular oxygen consumption and determined the basal oxygen consumption rate (OCR). After OCR was determined, oligomycin was injected to inhibit ATP synthase to cause a decrease in OCR. FCCP was then injected to increase electron flow through the electron transport chain. FCCP is an uncoupling agent that collapses the proton gradient and disrupts the mitochondrial membrane potential to cause maximal respiration. RRC was then calculated by taking the difference between maximal respiration and basal respiration, as depicted in the manufacturer’s profile (Fig. 5a). RRC measured the ability of the cell to respond to increased energy demand. The last injection was a combination of antimycin A and rotenone to shut down mitochondrial respiration.

The separation of compounds was performed with an Agilent Technologies 1290 Infinity LC-system equipped with a quaternary pump (G4204A), an auto sampler, HiP Sampler (G4226A), a column heater (G1316C). Instrument control and data analysis was carried out using Agilent MassHunter Workstation software version B.06. The chromatographic separation was performed using a Zorbax Eclipse Plus C₁₈ analytical column (Rapid Resolution HD, 2.1 X 50 mm, 1.8 μm) at 30 °C. The mobile phase consisted of methanol (Solvent B) and water with formic acid as solvent A (0.1 ml / 100 ml water). The flow rate was kept at 0.2 ml/min. The gradient elution started with 5%B - 95%B 0–10 min, 95%B 10–11 min, 95%B- 5%B 11–12 min and maintain 5%B for 12–15 min. The injection volume was 2 μl. Mass spectrometric analysis was performed on 6540UHD Accurate Mass Q-TOF LC/MS (G6540B). Mass spectra data was recorded on an ionization mode for a mass range of m/z 100–1700. Other mass spectrometer conditions were as follow: nebulizing gas pressure: 35 psi; drying gas flow: 8 L/min; drying gas temperature: 300 °C. The specific negative ionisation modes (m/z [M-H]⁻) were used to analyse the compounds.

The C2C12 cell line was used as a model system to investigate the effects of bilimbi leaf extract (DBB) on brown adipogenesis in this study. C2C12 is a murine myoblast which expressed Myf5, a myogenesis regulating protein during proliferation and skeletal muscle development. It is readily differentiated into mature myotubes in pro-myogenic medium culture, or developed into brown adipocytes upon appropriate stimulations. Rosiglitazone (ROSI), a PPARgamma agonist with known browning activities on both classic white adipocytes and inducible brown adipocytes, was served as the positive control. Adipogenesis was observed on day 4 after the initiation of differentiation (Additional file 1) by ROSI, similar phenomenon was found in DBB treated cells. Both treatments developed into fully mature adipocytes after 7 days of incubation. The development of adipocytes increased the intracellular accumulation of lipid droplets. These lipid droplets were then captured by Nile Red staining and imaged at 10X magnification (Fig. 1). Based on the cell morphology and lipid droplets accumulation, 1 μM ROSI, 100 μg/ml & 200 μg/ml DBB were demonstrated to induce adipocyte differentiation in C2C12 myoblasts. Myotubes development was also observed in DBB treated cells. Interestingly, these myoblasts were not found to undergo adipocyte differentiation in the presence of adipogenesis supplementation without DBB or ROSI stimulant. Nile Red staining did not capture lipid droplets in DMSO treated cells and was fully developed into myotubes.

Brown and brite adipocyte developments were anticipated upon DBB stimulation on both Myf5-positive and -negative progenitor cells. However, it was relatively difficult to identify between white, brown and beige adipocytes from the morphological appearance (Fig. 2). Further analysis was required to support the phenotypic results.

To anticipate the development of brite adipocytes upon stimulant, 3 T3-L1 preadipocyte was selected for this study. 3 T3-L1 is a non-Myf5 progenitor cell which only differentiates into white adipocyte upon maturity. Once fully differentiated white adipocytes were obtained, they were treated with either ROSI or DBB. These stimulants up-regulated the protein expressions of UCP1, PRDM16, FNDC5 and PGC1α in compared to DMSO treated cells, as shown in the fluorescent intensity signals captured by the IN Cell Analyzer image acquisition software (Fig. 3a). The captured intensities were then translated to quantifiable numbers. With DMSO treated cells assigned to the value of 1, signals produced from either ROSI or DBB treatments were normalised to this value (as fold increase in expression). As demonstrated in Fig. 3b, treatment with ROSI resulted in 2.6-fold increase in UCP1, 1.4-fold in PRDM16, 1.4-fold in FNDC5 and 3.7-fold in PGC1α protein expressions. Similar increments in the expression of these proteins were observed in 100 μg/ml DBB treated cells. DBB treatment on fully developed white adipocytes induced the protein expression of UCP1 and PRDM16 by 2.2-fold and 1.7-fold, FNDC by 3.2-fold and PGC-1α by 3.4-fold, significantly higher than non treated adipocytes. Treatment with DBB also showed 1.8-fold higher increment in FNDC5 than ROSI.

We then revisited the PRDM16 expression in the presence of DBB at increasing incubation period of time. PRDM16 is a known brown adipocyte determination factor which determines the thermogenic program of white adipocytes. Fully differentiated adipocytes were treated with increasing doses of DBB. Figure 4a demonstrated that treatment of 3 T3-L1 white adipocytes with ROSI could induce higher PRDM16 protein expression than the control, most remarkably observed after 7 to 10 days of treatment. Similarly, treatment with 25 μg/ml – 100 μg/ml DBB resulted in dose dependent increase in PRDM16 expression after 4, 7 and 10 days of treatments. The expression showed a decline in the 150 μg/ml DBB treatment group. The stimulation of PRDM16 expression was further validated with western blotting analysis as depicted in Fig. 4b, which detected the presence of PRDM16 in both cell types after 7 days of DBB treatment. 50 μg/ml - 100 μg/ml DBB treatments augmented PRDM16 protein expression in both cell types. A decline was observed in 200 μg/ml DBB treated cells.

To investigate whether elevation in brown adipocyte protein expression confer metabolism and functional effects, the mitochondria activity of adipocytes upon DBB treatment was measured. Oxygen consumption rate (OCR) of treated adipocytes was determined using a Seahorse XF24 extracellular flux analyzer as demonstrated in Fig. 5b & c. Basal OCR or basal metabolic rate (BMR) was significantly higher in DBB and ROSI compared to DMSO (vehicle) treated cells, suggesting higher adenoside triphosphate (ATP) turnover and demand upon DBB and ROSI treatment. Higher basal OCR may also indicate an increase in mitochondrial mass. Three steps were involved in the measurement of mitochondrial function. In step 1, an inhibitor of mitochondrial ATP synthase, oligomycin was added immediately after the BMR stabilised. The inhibition of ATP synthase reduced electron flow through the electron transport chain. The OCR level after oligomyin injection indicated proton leak, either through non-mitochondrial respiration or mitochondria uncoupling. Higher proton leak was observed in DBB and ROSI treated cells.

The injection of proton ionophore carbonyl cyanide 4-(trifluoromethoxy) phenylhydrozone (FCCP) in step 2 induced maximal substrate oxidation and causing maximum respiration. The stimulated OCR observed was due to uncoupling of electron transfer which allowed unrestricted electron flux through the electron transport chain to achieve maximal mitochondrial respiration capacity (MRC). MRC was also higher in both DBB and ROSI treated adipocytes. In step 3, the rotenone and antimycin A mixture shut down mitochondrial respiration to measure potential non-mitochondrial respiration that was driven by processes outside the mitochondria, and to determine the oxygen consumption via electron transport chain independent mechanisms. Reserve respiratory capacity (RRC) was significantly higher in DBB treated cells, indicating the potential to increase supply of energy demand during stress or increase in cellular activities.

DBB was shown to be non-toxic in all the experiment conducted. Our findings in cytotoxicity assay also supported that DBB is non-toxic to both cell lines at a test concentration as high as 200 μg/ml after 9 days of treatment (Additional file 2).

LC/MS studies were carried out on two batches of DBB ethanolic leaf extract with chromatograms as shown in Fig. 6a & b. The LC/MS analysis has enabled us to identify 18 predominant compounds as tabulated in Table 1.