Page 23 - N. Metabolism and metabolic diseases

Anti-obese mechanisms of anthocyanin-rich black soybean N-12
testa (ABST-3) in the beige-like adipocytes (BLA)

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Minjee Lee , Juhee Kim , Suyeon Lee , Dajeong Son , Myoungsook Lee 1,2*
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1 Research Institute of Obesity Sciences, Department of Food and Nutrition, Sungshin Women’s University (*corresponding author)
ABSTRACT
There were many studies that the effects of non-shivering thermogenesis and metabolic power of brown adipocytes (BAT) have been recognized. To find those effects of beige-like
adipocytes (BLA) on preventing obesity is challengeable. Using the extracts of anthocyanin-riched black soybean testa (ABST-3), the purpose of this study was to confirm energy
mechanisms of BLA on preventing obesity. ABST-3 dose-dependently reduced the lipid synthesis and accumulation in both WAT and BLA. Glycerol contents in the media and
phosphorylation of AMPKα as a lipolysis marker were increased by ABST-3, and the lipolysis effect was significantly higher in BLA than in WAT. The biomarkers of beiging from white
adipocytes, mitochondrial-RFP staining and oxygen consumption to find mitochondria activation were sharply increased in the adipocytes treated ABST-3 rather than the untreated one. They
were also higher in BLA than in WAT. In conclusion, ABST-3 activated mitochondria and facilitated the formation of BLA from WAT with mechanism of induction of thermogenesis and energy
consumption. With repeated scientific evidences, the ABST-3 can be recommended as a functional foods that has anti-obesity effects.
Key words : anthocyanin, brown adipocytes (BAT), beige-like adipocytes (BLA), thermogenesis
BACKGROUND METHODS
Obesity, a condition of excessive fat, effects metabolic diseases such as 1.Cell Culture : 3T3-L1 cells were cultured at 37°C and 5% CO 2 incubator. Until the cells were confluent, cells
cardiovascular disease and type 2 diabetes. According to the 2017 were maintained in Delbecco’s Modified Eagle’s Medium (DMEM) with 10% bovine calf serum and 1%
KNHANES statistics, the obesity prevalence has been increased in Korean penicillin/streptomycin. After post-confluency, cells were differentiated using MDI in medium with 10% fetal bovine
adults over 19 years old, exceeding 30% in 2005. Since Korean obesity serum (FBS) and then cultured in medium with insulin. Triiodothyronine (T 3 ) treated to induce the BLA model. BLA
tends to increase at all life cycles despite efforts to prevent obesity, the cells were maintained in medium with MDI, T 3 , and rosiglitazone. ABST-3 extracts (100, 200, 400 μg/mL) were
functional foods to prevent obesity have been developed in the industry added to the culture medium every other day during differentiation.
markets with the research evidence. In herbal medicine, anthocyanin-rich 2.Oil red O staining : Cells were fixed, washed, and stained with Oil Red O solution at RT. After washing the cells,
foods have been known to be beneficial to health. They show high lipid droplets were monitored under microscope.
antioxidant activity and are used to prevent heart disease, hypertension, 3.TG and free glycerol assay : Adipogenesis detection assay kit (Abcam, Cambridge, MA, USA) was used to
and obesity. In our previous study, anthocyanin-rich black soybean testa quantify TG concentrations. Free glycerol reagent (Sigma-Aldrich, St Louis, MO, USA) was used following the
(ABST-3) significantly inhibited lipogenesis genes and expressed the manufacturer’s protocol.
lipolysis genes. In the randomized clinical trial, ABST-3 reduced BMI and 4.Fluorescence microscopy : Cells were grown on coverslips during differentiation. Cells were washed, fixed and
waist circumferences and improved the plasma lipid profiles in obese stained with DAPI, BODIPY, and mitochondrial-RFP dye to observe the nuclear, lipid droplets, and mitochondria,
subjects. Two types of thermogenic fats, brown adipocytes (BAT) and respectively. Images were taken using a Confocal Laser Scanning Microscope.
beige-like adipocyts (BLA) possess both common and distinct 5.Western blotting analysis : The harvested cells were lysed and the total proteins were qualified. The proteins
characteristics that included metabolikc regulation and function. BLA has a separated from 12% of SDS-PAGE gel were transferred to NC membrane. The membrane was blocked using
unique gene that is differente from BAT and expresses thermogenic genes TBS-T containing 3% BSA for 3 h at RT. After reacting the primary antibodies and secondary antibodies, proteins
by stimulators. Therfore, the developmental process of BLA will be a were visualized by Bio-Rad chemiluminescence system.
unique model for the new theraqeutic target in preventing obesity. The 6.Energy and oxygen consumption rate (OCR) analysis : The NAD/NADH ratio were evaluated with a
purpose of our study is identify whether ABST-3 extracts activate the NAD/NADH quantification colorimetric kit (Biovision Inc., Mountain View, CA, USA). MitoXpress Xtra Oxygen
energy metabolism including thermogenesis in T 3 –induced BLA to prevent consumption assay kit (Agilent, California, USA) and seahorse XFe24 extracellular analyzer were used to
obesity. measure oxygen consumption. Measurement was followed the manufacturer’s protocol.
RESULTS
A B A B

C D V C D

Figure 1. Inhibitory effects of ABS extracts on TG accumulation in WAT and BLA. (A) Figure 2. Comparison of transcription factors about the effects of ABS extracts on adipogenesis, lipolysis,
Cells were treated with/without ABS extracts up to 500 μg/mL and cell viability was measured thermogenesis, and mitochondrial function in WAT and BLA. (A) Adipogenic protein expression of PPARγ was
by MTT assay. (B) Lipid droplets of cells stained with ORO solution was extracted and determined by western blotting. (B) Lipolytic protein expression of AMPKα and pAMPKα were analyzed by western blotting,
quantified at OD 490-520 nm in WAT and BLA. (C) Intracellular TG contents were determined and glycerol secretion was measured in a medium. (C) Thermogenic protein of UCP1 was determined by western blotting. (D)
by the enzyme assay at 570 nm in WAT and BLA. (D) The nuclear and lipid droplets were Protein expression of cytochrome C related to mitochondrial function was determined by western blotting. Each bar
detected by DAPI and BODIPY staining. Each bar represents the mean±S.D. in triplicate represents the mean±S.D. in triplicate experiment. Data were analyzed by one-way analysis of ANOVA with Duncan’s
experiment. Data were analyzed by one-way analysis of ANOVA with Duncan’s test(NS, not test(NS, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). Small letters (a, b, c, d) represent significant
significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). Small letters (a, b, c, d) differences between groups at p<0.05.
represent significant differences between groups at p<0.05.
A B C

Figure 3. Effects of ABS extracts on mitochondrial activation and energy and oxygen consumption in WAT and BLA.
(A) The nuclear and mitochondria were stained by DAPI and mitochondrial-RFP dye. (B) The NAD/NADH ratio was measured at 450 nm by the quantification colorimetric kit. (C) OCR recorded every 17 seconds was
measured at 380 and 650 nm and OCR in BLA was determined by seahorse XFe24 extracellular analyzer. Each bar represents the mean±S.D. in triplicate experiment. Data were analyzed by one-way analysis of
ANOVA with Duncan’s test(NS, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). Small letters (a, b, c, d) represent significant differences between groups at p<0.05.
CONCLUSION
1. ABS extracts contribute to the potentiality of the energy balance in body by reducing fat accumulation and increasing lipolysis and oxygen consumption via mitochondrial activation.
2. Anthocyanins can be developed as good functional materials to control the energy metabolism on the obesity prevalence in the future.
ACKNOWLEDGEMENTS
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea Government (MSIP; 2019R1A2C1008434) and “Cooperative Research Program for
Agriculture Science and Technology Development (No.PJ015269012020)” Rural Development Administration, Republic of Korea.
REFERENCES
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Food 2015, 14, 623-633.
2.Giralt, M.; Villarroya, F. White, brown, beige/brite: different adipose cells for different functions? Endocrinology. 2013, 154(9), 2992-3000.