KR102606901B1 - A pharmaceutical composition for prevention or treatment of obesity comprising biochanin a or pharmaceutically accepted salts thereof as an effective component - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of obesity containing Biochanin A (BioA) or a pharmaceutically acceptable salt thereof, and more specifically, to a pharmaceutical composition for the prevention or treatment of obesity in subjects. Or pharmaceutical use for preventing or treating obesity in the subject by differentiating mesenchymal stem cells (MSCs) in the subject into brown adipose cells by treating a pharmaceutically acceptable salt thereof. It is about etc. When BioA of the present invention or a pharmaceutically acceptable salt thereof is treated with an object, it can effectively induce differentiation of adipose tissue within the object into brown fat, so it can be effectively used for obesity and diseases related thereto, making it suitable for use in the pharmaceutical, food and cosmetics industries. It's a very useful invention.

Description

Pharmaceutical composition for preventing or treating obesity comprising biochanin A or a pharmaceutically acceptable salt thereof

The present invention relates to a pharmaceutical composition for the prevention or treatment of obesity containing Biochanin A (BioA) or a pharmaceutically acceptable salt thereof, and more specifically, to a pharmaceutical composition for the prevention or treatment of obesity in subjects. Or pharmaceutical use for preventing or treating obesity in the subject by differentiating mesenchymal stem cells (MSCs) in the subject into brown adipose cells by treating a pharmaceutically acceptable salt thereof. It is about etc.

Adipose tissue regulates whole body energy metabolism, from nutrient intake and excess energy storage to consumption (Choe, Huh, Hwang, Kim, & Kim, 2016). In mammals, energy homeostasis is controlled by two types of adipose tissue (white and brown), each with unique morphology and function. Brown adipose tissue (BAT) is characterized by a unique thermogenic system containing densely packed eccentric mitochondria that release excess energy as heat. BAT contains pleiotropic cytoplasmic lipid droplets and continuously generates heat by oxidizing free fatty acids (FFA) within adipocytes rather than supplying them to other cells. In contrast, white adipose tissue (WAT) is characterized by an excellent storage capacity for excess energy in the form of fat (Coelho, Oliveira, & Fernandes, 2013). Nevertheless, extensive studies have also identified a new type of thermogenic cells known as beige cells, which reside in WAT but have thermogenic functions similar to brown adipocytes, including large amounts of mitochondria (Coelho et al., 2013; Kajimura , Spiegelman, & Seale, 2015).

Fat dysfunction is caused by an imbalance between dietary energy intake and energy expenditure, leading to obesity and related disorders such as insulin resistance, dyslipidemia, hepatic steatosis, and type 2 diabetes (Jung & Choi, 2014; Longo et al. ., 2019). These metabolic problems are mainly caused by dysregulation of mitochondrial biogenesis and oxidative phosphorylation (OXPHOS) and altered lipid profile due to WAT dysfunction (Chouchani & Kajimura, 2019). To solve the dilemma of adipose dysfunction, induction of BAT function, differentiation of mesenchymal stem cells (MSCs) into brown-fat-like (i.e. brown and beige) adipocytes, or white fat Several therapeutic approaches have been known, such as transdifferentiation of cells into beige adipocytes (a process known as browning). Several transcription factors are known to be involved in adipocyte differentiation and adipocyte fate during brown or beige adipogenesis. PPARγ, a master regulator of adipogenesis, determines adipocyte activity, while C/EBP maintains PPARγ expression and acts synergistically with PPARγ to promote the expression of genes active in both brown and white adipocytes (Uldry et al., 2006). Additionally, PPARγ stabilizes Prdm16 expression, which regulates mitochondrial biogenesis and cellular respiration by inducing the expression of PGC1α, UCP1, and DIO2 in WAT (Becerril et al., 2013; Farmer, 2006; Seale et al., 2011).

Mitochondrion production plays an important role in thermogenesis, is controlled by PGC1α, a PPARγ coactivator, and can activate thermogenesis through upregulation of the brown adipocyte signature (Farmer, 2006 ; Sarjeant & Stephens, 2012). In response to specific external signals, PGC1α activates an adaptive thermogenic program in brown fat, including increased fuel intake, fatty acid oxidation, mitochondrial production, OXPHOS, and oxygen consumption (Uldry et al., 2006). Mitochondrion production leads directly to lipolysis, which can supply FFA for mitochondrial β-oxidation. Lipolysis is initiated by adipose triglyceride lipase (ATGL), is required for maximal induction of thermogenic genes, and increases mitochondrial biogenesis by supplying FFA for mitochondrial β-oxidation (Kershaw et al., 2007).

Various classes of phytochemicals have been reported to promote brown fat-like adipocyte formation and mitochondrial production (Hong et al., 2019; Imran et al., 2017; Liu et al., 2019; Qi et al., 2019; Rahman & Kim, 2020b; Rasbach & Schnellmann, 2008; Shen, Liu, Ng, Chan, & Yong, 2006; Velickovic et al., 2019; Wang et al., 2018; You et al., 2015; Zhang et al., 2019).

Biochanin A (BioA), an O-methylated isoflavone present in red clover, alfalfa, cabbage and many other plants, is known to have lipid-lowering, anticancer and anti-inflammatory activities (Cassady et al., 1988; Oza & Kulkarni , 2018). BioA has been reported to act as a PPARγ and SIRT1 agonist and induce adipogenesis in 3T3-L1 preadipocytes (Oza & Kulkarni, 2018; Shen et al., 2006).

However, to date, the effect of BioA on brown adipocyte formation has not been revealed.

The problem to be solved by the present invention is to provide a pharmaceutical composition for effectively preventing and treating obesity in a subject by differentiating adipose tissue into brown fat by treating the subject with BioA or a pharmaceutically acceptable salt thereof. will be.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating obesity containing Biochanin A or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a health functional food for preventing or improving obesity containing Biochanin A or a pharmaceutically acceptable salt thereof as an active ingredient.

Additionally, the present invention provides a cosmetic composition for slimming containing Biochanin A or a pharmaceutically acceptable salt thereof.

The cosmetic composition is preferably in a formulation selected from the group consisting of lotion, lotion, cream, essence, foam, and pack.

Additionally, the present invention provides a composition for inducing differentiation of mesenchymal stem cells into brown adipocytes, comprising Biochanin A or a pharmaceutically acceptable salt thereof as an active ingredient.

The differentiation induction is preferably by a mechanism selected from the group consisting of reducing intracellular triglyceride accumulation, inducing lipolysis, increasing the number of intracellular mitochondria, inducing a brown fat phenotype in cells, and activating AMPK signaling.

Additionally, the present invention provides a method of inducing differentiation into brown adipocytes, comprising treating mesenchymal stem cells isolated from the human body with Biochanin A or a pharmaceutically acceptable salt thereof.

When BioA of the present invention or a pharmaceutically acceptable salt thereof is treated with an object, it can effectively induce differentiation of adipose tissue within the object into brown fat, so it can be effectively used for obesity and diseases related thereto, making it suitable for use in the pharmaceutical, food and cosmetics industries. It's a very useful invention.

Figure 1 shows the effect on cell viability (and TG accumulation) of C3H10T1/2 MSCs by BioA treatment.
Figure 2 shows the effect of lipolysis of C3H10T1/2 MSCs by BioA treatment.
Figure 3 shows the effect of mitochondrial production in C3H10T1/2 MSCs by BioA treatment.
Figure 4 shows the expression patterns of thermogenic genes, adipogenic genes, beige-fat marker genes, brown-fat marker genes, and white-fat marker genes in C3H10T1/2 MSCs by BioA treatment.
Figure 5 shows the effect of AMPK signaling pathway in C3H10T1/2 MSCs by BioA treatment.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention investigated the effects of biochanin A (BioA), an O-methylated isoflavone, on brown fat phenotype formation and related thermogenic programs, including mitochondrial production and lipolysis, in C3H10T1/2 MSCs. Our data show that treatment with BioA in the adipogenic differentiation cocktail induced the formation of brown fat-like adipocytes from C3H10T1/2 MSCs even without treatment of known browning inducers (rosiglitazone or T3) in the early stages of differentiation. The formation of brown fat-like adipocytes by BioA treatment was evidenced by the upregulation of key thermogenic markers Ucp1, Pgc1α, Prdm16, and Pparγ. Additionally, BioA treatment promoted mitochondrial biogenesis as judged by upregulation of Cox8b, Cidea, Dio2, Sirt1, Opa1, and Fis1 genes. BioA treatment increased the amount of mitochondrial DNA and its encoded proteins, oxidative phosphorylation complexes (I-V), and these changes were associated with high oxygen consumption by C3H10T1/2 MSCs. Small interfering RNA-induced gene knockdown and dosomorphine-induced competitive inhibition experiments showed that BioA exerts its thermogenic action through activation of AMPK signaling. Our study reveals the mechanism by which the brown fat phenotype is promoted by BioA. Nevertheless, additional clinical studies are needed to confirm that BioA is a brown fat-like phenotype inducer.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating obesity containing Biochanin A or a pharmaceutically acceptable salt thereof as an active ingredient.

The term “obesity” refers to a clinical condition in which the amount of fat in an individual exceeds the normal range. Additionally, the obesity includes metabolic syndrome caused by various metabolic abnormalities related to obesity.

The pharmaceutical composition containing the active ingredient of the present invention can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., and injections of sterile injectable solutions according to conventional methods to suit each purpose of use. It can be formulated and used in various forms, and can be administered through various routes, including oral administration, intravenous, intraperitoneal, subcutaneous, rectal, and local administration.

These pharmaceutical compositions may additionally contain carriers, excipients or diluents, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. etc. can be mentioned. In addition, the pharmaceutical composition of the present invention may further include fillers, anti-coagulants, lubricants, wetting agents, flavorings, emulsifiers, preservatives, etc.

In a preferred embodiment, solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient in the pharmaceutical composition, such as starch, calcium carbonate, It is formulated by mixing sucrose, lactose, gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate, talc, etc. may be used.

As a preferred embodiment, oral liquid preparations include suspensions, oral solutions, emulsions, syrups, etc., and in addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, Fragrances, preservatives, etc. may be included.

As a preferred embodiment, preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, etc. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Injectables may contain conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, etc.

The active ingredient of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, activity of the drug, and the type of patient's disease. It can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the field of medicine. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

In a preferred embodiment, the effective amount of the active ingredient in the pharmaceutical composition of the present invention may vary depending on the patient's age, gender, and weight, and is generally 1 to 5,000 mg per kg of body weight, preferably 100 to 3,000 mg per day. Alternatively, it can be administered every other day or divided into 1 to 3 doses per day. However, since it may increase or decrease depending on the route of administration, severity of disease, gender, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered to a subject through various routes. All modes of administration are contemplated, for example, oral, rectal or by intravenous, intramuscular, subcutaneous, intrathecal or intracerebroventricular injection.

In the present invention, "administration" means providing a predetermined substance to a patient by any appropriate method, and the route of administration of the pharmaceutical composition of the present invention is oral or parenteral through all general routes as long as it can reach the target tissue. It can be administered orally. Additionally, the composition of the present invention may be administered using any device capable of delivering the active ingredient to target cells.

In the present invention, “subject” includes, but is not particularly limited to, for example, humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs. And, preferably, it means mammals, and more preferably, humans.

In addition, the present invention provides a health functional food for preventing or improving obesity containing Biochanin A or a pharmaceutically acceptable salt thereof as an active ingredient.

The health functional food of the present invention can be used in a variety of foods and beverages that are effective in preventing or improving various symptoms related to obesity. Foods containing the active ingredient of the present invention include, for example, various foods, beverages, gums, teas, vitamin complexes, health supplements, etc., and can be used in the form of powders, granules, tablets, capsules, or beverages. .

The active ingredient of the present invention can generally be added at 0.01 to 15% by weight of the total food weight, and the health drink composition can be added at a rate of 0.02 to 10g, preferably 0.3 to 1g, based on 100ml.

In addition to containing the above compounds as essential ingredients in the indicated ratio, the health functional food of the present invention may contain foodologically acceptable food additives, such as natural carbohydrates and various flavoring agents, as additional ingredients.

Examples of the natural carbohydrates include common sugars such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.

As the flavoring agent, natural flavoring agents such as thaumatin, rebaudioside A, or stevia such as glycyrrhizin, and synthetic flavoring agents such as saccharin and aspartame may be used. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g, per 100 ml of the health functional food of the present invention. In addition to the above, the health functional food of the present invention contains various nutrients, vitamins, minerals, flavoring agents such as synthetic and natural flavors, colorants and thickening agents, pectic acid and its salts, alginic acid and its salts, organic acids, and protective colloids. It may contain thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the health functional food of the present invention may contain pulp for the production of natural fruit juice, fruit juice drinks, vegetable drinks, etc. These ingredients can be used independently or in combination. The ratio of these additives is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the active ingredient of the present invention.

Additionally, the present invention provides a cosmetic composition for slimming containing Biochanin A or a pharmaceutically acceptable salt thereof.

The term “slimming” refers to a phenomenon in which the volume of the entire body or a specific area is reduced. For example, it may be the removal or reduction of fat in the entire body or a specific area.

The active ingredient of the cosmetic composition of the present invention may be included in an amount of 0.01 to 50% by weight, preferably 0.1 to 20% by weight, and more preferably 1 to 10% by weight, based on the total weight of the composition. Within this content range, appropriate formulation stability can be secured and the desired slimming effect can be expected.

In addition to the active ingredients of the present invention, the composition of the present invention can be used by additionally containing known natural extracts or other ingredients for antioxidant, anti-aging, moisturizing, and skin regeneration promotion effects to the extent that they do not impair the effective activity of the present invention. You can.

In addition, the composition may contain ingredients commonly added to cosmetic compositions, such as fatty substances, organic solvents, solubilizers, thickeners, gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, Cosmetic or cosmetic applications such as fragrances, surfactants, water, ionic or non-ionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic activators. It can be molded into a specific formulation by including adjuvants or carriers commonly used in the field of dermatology.

The cosmetic composition of the present invention can be prepared in any formulation commonly prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, pack, soap, surfactant- It can be formulated as a cleansing agent, oil, spray, etc., but is not limited to this. More specifically, lotion, lotion, softening lotion, nourishing lotion, cream, nourishing cream, massage cream, essence, eye cream, powder foundation, emulsion foundation, wax foundation, cleansing cream, foam, cleansing foam, cleansing water, pack. , spray, powder, pact, lip gloss, lipstick, shadow, shampoo, and rinse.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier ingredient. You can.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as the carrier ingredient. In particular, when the formulation is a spray, chlorofluorohydrocarbon, May contain propellants such as propane/butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent, or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, These include 1,3-butyl glycol oil, glycerol aliphatic esters, polyethylene glycol or fatty acid esters of sorbitan.

When the formulation of the present invention is a suspension, the carrier ingredients include water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, and microcrystals. Cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used.

When the formulation of the present invention is a surfactant-containing cleansing agent, the carrier ingredients include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, and fatty acid amide. Ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester can be used.

Additionally, the present invention provides a composition for inducing differentiation of mesenchymal stem cells into brown adipocytes, comprising Biochanin A or a pharmaceutically acceptable salt thereof as an active ingredient.

The differentiation induction is preferably by a mechanism selected from the group consisting of reducing intracellular triglyceride accumulation, inducing lipolysis, increasing the number of intracellular mitochondria, inducing a brown fat phenotype in cells, and activating AMPK signaling.

The composition for inducing differentiation may be applied, for example, as a reagent composition for research. The composition for inducing differentiation of the present invention can be administered to cells or experimental animals under normal laboratory conditions, and it is preferable that specific administration methods and safety rules are carried out in accordance with the internal compliance regulations of each experimenter's laboratory.

Additionally, the present invention provides a method of inducing differentiation into brown adipocytes, comprising treating mesenchymal stem cells isolated from the human body with Biochanin A or a pharmaceutically acceptable salt thereof.

The mesenchymal stem cells are those isolated from an individual by natural or artificial methods. The cells isolated from an individual can be cultured for several days to several weeks under appropriate culture conditions, and in some cases, may be stored frozen.

The method of administering the active ingredient of the present invention to the separated cells is obvious to those skilled in the art, and can preferably be followed as described in the examples below in this specification.

Hereinafter, the present invention will be described in more detail through specific examples. The following examples describe a preferred embodiment of the present invention, and it is clear that the scope of the present invention is not to be construed as limited by the matters described in the following examples.

[ Example ]

1. Materials and methods

1.1. Chemicals, Reagents and Antibodies

BioA (Figure 1A; #D2016), insulin, dexamethasone, IBMX, rosiglitazone (Rosi), dosomorphine (DRS), Oil Red O dye (ORO), 3-(4,5-dimethylthiazol-2-yl)- 2,5-Dimethyltetrazolium bromide (MTT), 4% formamide, mimethyl sulfoxide, and 40,6-diamidino-2-phenylindole (DAPI) were from Sigma-Aldrich (St. Louis, MO). Carbonyl cyanide p-trifluoro-methoxyphenyl hydrazine (FCCP) was purchased from Cayman Chemicals (Ann Arbor, MI). Fetal bovine serum (FBS) and high-glucose Dulbecco's modified Eagle's medium (DMEM) were purchased from Atlas Biologicals (Fort Collins, CO), and penicillin-streptomycin solution was purchased from Hyclone Laboratories Inc. (South Logan, NY). ) was purchased from Antibodies against PGC1α, PRDM16, and OXPHOS proteins were purchased from Abcam (Cambridge, MA). HSL, ATGL, phospho-(p-)HSL (Ser563 and Ser660), Sirt1, aP2, C/EBPβ, AMPK, p-AMPK (Thr172), p38 MAPK, p-p38 MAPK (Thr180/Tyr182), and p-PKA. Antibodies to the substrate and anti-rabbit IgG (H + L), F(ab')2 fragment antibody (Alexa Fluor 488 conjugate), anti-rabbit IgG antibody conjugated to horseradish peroxidase, horseradish peroxidase Conjugated anti-mouse IgG antibodies, and MitoTracker ^® Red CMXRos were purchased from Cell Signaling Technology (Danvers, MA), while UCP1, PPARγ, and β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX). The BCA protein assay kit was obtained from Thermo Fisher Scientific (Rockford, IL), and protein loading buffer was obtained from Bio-Rad Laboratories, Inc. (Hercules, CA).

1.2. Cell culture, differentiation, and processing

C3H10T1/2 MSC (KCLB-10226; passage number ≥ 10) were cultured in DMEM GlutaMax supplemented with 10% FBS and 1% penicillin-streptomycin solution in a humidified 5% CO ₂ incubator at 37°C. For adipogenic differentiation, fully grown C3H10T1/2 cells (2 days after confluency, designated as day 0, Figure 1B) were incubated with the adipogenic differentiation cocktail, i.e., in the presence or absence of BioA in DMEM supplemented with 10% FBS. , incubated with MDI (0.5 mM IBMX, 1 μM dexamethasone, and 10 μg/mL insulin). Treatments exceeding 2 days were continued until the 4th day. In this case, we mixed BioA with maturation medium containing DMEM, insulin, and 10% FBS for cell culture on days 3 and 4. After 4 days, only the maturation medium was used until harvest. Fully differentiated C3H10T1/2 MSCs were harvested on day 6 and used in this study. Culture in MDI was used as a negative control, and culture with Rosi (1 μM) in DMEM medium was used as a positive control.

1.3. Cell viability analysis

C3H10T1/2 MSCs were seeded in 96-well plates at a density of 80% to 90% confluency the next day and incubated with 10, 20, or 40 μM BioA for 24, 48, or 72 hours. . Afterwards, 20 μL of 5 mg/mL MTT solution was added to each well containing 200 μL of culture medium, and the cells were cultured at 37°C for 4 hours to allow formazan crystals to form. Afterwards, formazan crystals were dissolved in 150 μL of dimethyl sulfoxide and absorbance was measured at a wavelength of 590 nm using a Victor™ X3 multi-label reader (Perkin Elmer, Waltham, MA).

1.4. ORO staining analysis

Total triglyceride (TG) content was measured by ORO staining. C3H10T1/2 MSCs were differentiated for 6 days in 6-well plates with MDI with or without BioA as described above. Mature cells were washed twice with 1x phosphate-buffered saline (PBS), fixed with 10% formalin for 1 hour, and then stained with filtered 0.3% ORO solution for 20 minutes. Stained cells were washed four times with distilled water, and phenotypic changes were photographed using an Axiovert-25 microscope (Carl Zeiss, Jena, Germany). To quantify ORO, the dye was eluted from stained cells using 100% isopropanol, and absorbance was measured at 520 nm on a Victor™ X3 instrument (Perkin Elmer).

1.5. Gene expression analysis

Undifferentiated and differentiated C3H10T1/2 MSCs were harvested, total RNA was extracted using an RNA extraction kit (Qiagen, Valencia, CA), and total RNA concentration was determined on a Scandrop spectrophotometer (Analytik Jena AG; Jena, Germany). Measured.

cDNA was synthesized by reverse transcription PCR (RTPCR) of a total of 1 μg of RNA using the Maxime RT PreMix Kit (Intron Biotechnology, Seoul, Korea), and the reaction was performed in a Veriti 96-Well Thermal Cycler (Applied Biosystems, Singapore). did. Quantitative PCR (qPCR) was performed using the iQTM SYBR Green Supermix Kit (Bio-Rad, Singapore) on a CFX96™ Real-Time PCR detection system (Bio-Rad, Singapore), using Tbp as an internal control gene. did. The sequences of the primers are listed in Table 1 (SEQ ID NO: 1 to SEQ ID NO: 88).

[Table 1] Primer sequences

1.6. Western blot analysis

Preadipocyte samples and differentiated adipocyte samples were washed twice with ice-cold 1x PBS and incubated with protease inhibitor cocktail, 2 mM phenyl methylsulfonyl fluoride, 1 mM sodium orthovanadate (Santa Cruz Biotechnology), and phosphatase inhibitor cocktail ( It was dissolved in RIPA lysis buffer containing Sigma-Aldrich (Sigma-Aldrich). The lysate was scraped, incubated on ice for 15 min, and the supernatant was collected by centrifugation at 14,000 × g for 15 min at 4 °C. Total protein concentration was measured using the BCA protein assay kit. For each sample, equal amounts of total protein were loaded and separated on a sodium dodecyl sulfate 4-20% polyacrylamide gradient gel (Mini-PROTEAN Precast Gel, Bio-Rad). Afterwards, the protein was transferred to a polyvinylidene difluoride membrane (Trans-Blot SD Semi-Dry Cell, Bio-Rad) in a semi-dry transfer cell (Bio-Rad) at 13 V for 1.2 hours. The membrane was then blocked with 5% dry skim milk dissolved in 1x Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBST) for 1 h at room temperature on a shaker and then incubated with primary antibody overnight at 4 °C. was cultured with. Afterwards, the membrane was washed three times for 5 minutes at room temperature. Polyvinylidene difluoride membranes were incubated with specific horseradish peroxidase-conjugated secondary antibodies diluted in TBST for 1.5 h at room temperature on a shaker and then washed three times for 5 min each. All washing steps were performed in TBST. Immunoreactive protein signals were detected by chemiluminescence ECL analysis (Advansta), and images were acquired using molecular imaging software ImageLab 2.0 (Bio-Rad). Brightness and contrast were slightly adjusted for better visualization of the data, but the same changes were made to the entire image panel and no information was lost from any of the images. Staining with anti-β-actin antibody was used as a loading control for each protein expression analysis.

1.7. Mitochondrial content analysis

C3H10T1/2 MSCs were cultured in confocal dishes and differentiated for 6 days. Cell monolayers were then washed twice with 1x PBS and incubated with MitoTracker dye (diluted to a concentration of 100 nM in growth medium) for 15 min at 37 °C. Afterwards, cells were fixed in ice-cold methanol for 15 min at -20 °C and washed three times with PBS for 5 min each. Next, cells were permeabilized with 0.1% Triton X-100 in 3% BSA for 15 min and blocked with 1% BSA, 5% goat serum, and 0.3% Triton did. Cells were then incubated with primary antibody against Ucp1 (Abcam) overnight at 4°C, followed by Alexa Fluor 488-conjugated anti-rabbit IgG (secondary) antibody (Cell Signaling Technology) for 1 hour at room temperature. cultured for a while. For DNA staining, cells were incubated with 1 μg/mL DAPI for 1 min at room temperature and then washed three times with 1x PBS containing 0.25% Tween 20. The coverslip was mounted with a fluorescent mounting medium containing an anti-fading agent (Dako, CA). Images were captured using a confocal microscope (Olympus, Tokyo, Japan) and analyzed in FV10i-ASW 3.0 viewer software. Mitochondrial content was assessed as the ratio of nuclear DNA (nDNA) copy number to mitochondrial DNA (mtDNA) copy number quantified by qPCR, under the assumption that nDNA copy number is constant. For each DNA extract, the nuclear gene, ribosomal protein p0, and the mitochondrial gene cytochrome c oxidase subunit I (CoxI) were quantified individually by qPCR. Data were normalized to nuclear gene p0 DNA.

1.8. Ampkα knockdown

siAmpkα (Santa Cruz Biotechnology, Inc.), a small interfering RNA (siRNA) oligonucleotide duplex, was transfected into C3H10T1/2 MSCs using Lipofectamine 2000 at 80% to 90% confluency. That is, 100 pM siRNA was mixed with 6 μL of Lipofectamine 2000 per well and transfected into cells in a 6-well plate. Cells were briefly washed with 2 mL of Opti-MEM medium and 800 μL of this medium was added to each well. siRNA and Lipofectamine 2000 were individually diluted in 100 μL (per well) of Opti-MEM medium, incubated for 5 min, mixed, incubated for 30 min at room temperature, and then 200 μL per well into the designated wells of a 6-well plate. added to After 8 hours, 1 mL of growth medium was added to each well and incubated overnight at 37°C. The medium was replaced with induction medium 2 days after confluency, with or without BioA. The efficiency of siRNA-based knockdown was determined by qRT-PCR using Ampkα primers (Santa Cruz Biotechnology). Protein and RNA samples were prepared on day 6 of differentiation, and Western blotting and qRT-PCR were performed to analyze the expression of downstream genes.

1.9. Oxygen Consumption Analysis

C3H10T1/2 MSCs were seeded in 96-well plates at a density of approximately 4 × 10 ⁴ to 6 × 10 ⁴ cells per well in 200 μL of culture medium and maintained overnight in a CO ₂ incubator at 37°C. Six days after differentiation, FAO-driven oxygen consumption analysis was performed using the fatty acid oxidation complete assay kit (Abcam, MA) according to the manufacturer's instructions. The mitochondrial OXPHOS uncoupler FCCP was used as a positive control, and Etomoxir (CPT1α inhibitor) was used as a negative control. Readings were performed on a Victor™ X3 multilabel reader (Perkin Elmer) at excitation and emission wavelengths of 380 and 650 nm, respectively.

1.10. Mitochondrial membrane potential analysis

Mitochondrial membrane potential was measured using the benzimidazolecarbocyanine iodide (JC-1) mitochondrial membrane potential analysis kit (Abcam). Cells were incubated with JC-1 staining solution (Abcam) for 30 minutes at 37°C in the dark, and fluorescence was measured at 535 nm (excitation)/590 nm (emission) on a Victor™ measured (Tak et al., 2014).

1.11. Transmission electron microscopy analysis

Sections of differentiated C3H10T1/2 MSCs were fixed in 2.5% glutaraldehyde in 100 mM phosphate buffer (pH 7.2) overnight at 4 °C. The sections were then postfixed in 1% osmium tetroxide, dehydrated in increasing concentrations of ethanol, and embedded in fresh epoxy resin 618. Ultrathin sections were prepared and stained with lead citrate before testing. The instruments used in this experiment were TEM H-7000 (Hitachi, Japan) and Ultra microtome Ultracut-S (Lica, Germany) (Bartelt et al., 2011).

1.12. Density measurements in ImageJ

Immunoblot and immunofluorescence staining images were quantified in ImageJ software (NIH, Bethesda, MD). Before quantification, immunofluorescence images were converted to a weighted 8-bit grayscale format and then inverted.

1.13. statistical analysis

All values were expressed as mean ± SE of the mean (SEM) of triplicate biological samples. Depending on the experiment, ANOVA with student's t-test or Dunnett's multiple comparison test was performed to determine the significance of differences according to the following probabilities: < 0.05 (indicated by * in the figure), < 0.01 (**), < 0.001 (***) and > 0.05 (ns; not significant). All statistical analyzes were performed using GraphPad Prism 8 (GraphPad Software, La Jolla, CA).

2. Results

2.1. BioA improves intracellular TG accumulation in C3H10T1/2 MSCs

To find the therapeutic dose of BioA, we investigated the cell viability of three doses of BioA (10, 20, and 40 μM) for 24, 48, and 72 h via MTT assay. As shown in Figure 1C, treatment with ≤40 μM BioA had no noticeable cytotoxic effect, but rather improved cell viability. Additionally, we measured the effect of BioA on intracellular TG accumulation by ORO staining. As shown in Figure 1D, cells treated with differentiation medium (MDI) exhibited TG accumulation compared to the preadipocyte population (untreated control). In contrast, cells treated with rosiglitazone (Rosi; a PPARγ agonist) showed greater TG accumulation than control cells treated with MDI. Cells treated with BioA showed TG accumulation, but TG content was reduced by BioA in a dose-dependent manner. Treatment with 40 μM BioA reduced TG accumulation compared to cells treated with Rosi. Based on these results, it was decided to use 40 μM BioA for further experiments.

2.2. BioA induces lipolysis in C3H10T1/2 MSCs

We hypothesized that BioA may contribute to lipolysis. Therefore, we measured the expression of the key lipolysis-related genes, Hsl and Atgl. As illustrated in Figures 2A and 2B, BioA treatment significantly enhanced the expression of Hsl and Atgl (up to 11.4-fold and 7.0-fold, respectively) and caused phosphorylation of HSL at S660 and S563. Additionally, BioA treatment increased the expression of other lipolytic genes, including Mgll (approximately 2.4-fold) and Abhd5 (19.2-fold). For reference, BioA treatment enhanced the expression of the mitochondrial β-oxidation-related gene (Lcad; up to 2.7-fold) and the expression of the β3-AR gene (Adrb3), while upregulating the adipogenesis-related genes Acc (0.1-fold) and Dgat2 (0.2-fold). It was comprehensively downregulated. Next, to investigate morphological changes caused by BioA, we performed transmission electron microscopy of fully differentiated C3H10T1/2 MSCs. As shown in the electron micrograph in Figure 2C, BioA-treated cells contained smaller lipid droplets compared to MDI- and Rosi-treated cells. From these data, it could be inferred that BioA induces lipolysis while promoting mitochondrial β-oxidation in differentiated C3H10T1/2 MSCs.

2.3. BioA promotes mitochondrial biogenesis and respiration in C3H10T1/2 MSCs

To assess the effect of BioA on mitochondrial properties, we examined the expression of mitochondria-biogenesis-related genes. BioA treatment significantly increased the levels of Sirt1 mRNA (up to 9.2-fold) and protein compared to MDI- and Rosi-treated cells (4.5-fold, Figure 3A and 3D). Additionally, we observed that BioA treatment slightly improved NAD ⁺ homeostasis and SIRT1 activity. Additionally, BioA slightly upregulated other mitochondria-biogenesis-related genes (i.e., Cox8b, Cidea, and Dio2) and mitofusion-fission-related genes Opa1 and Fis1 compared to MDI-treated cells (Figure 3A). To determine the effect of BioA on mitochondrial biogenesis, we performed immunofluorescence staining of mitochondrial contents: mtDNA with DAPI, mitochondria with MitoTracker red, and uncoupling protein 1 (UCP1) protein by immunoblotting. . As shown in Figure 3B, BioA treatment increased mitochondrial content and expression of mitochondrial UCP1. As a result of quantifying the expression of the mtDNA gene (Cox1), it was confirmed that BioA treatment increased mtDNA copy number compared to MDI-treated and Rosi-treated cells (Figure 3C). Next, we investigated the effects of BioA on mitochondrial respiratory capacity and health. To understand the effect of BioA on mitochondrial respiration, we first quantified the mtDNA-encoded OXPHOS proteins (complexes I to V: CI to CV), which are embedded in the major electron transport chain. As shown in Figure 3D, BioA treatment enhanced OXPHOS and ATP production as judged by high expression of OXPHOS complexes I to V. Nevertheless, changes in the expression of ATP synthase (CV) were not clearly visible, probably due to the hydrophobic nature of the OXPHOS protein. To determine OXPHOS activity, we analyzed the oxygen consumption rate by fatty acid oxidation associated with the expression of OXPHOS proteins. As illustrated in Figure 3E , BioA treatment increased oxygen consumption compared to MDI- and Etomoxir (CPT1α inhibitor; negative control)-treated cells. In contrast, oxygen consumption rates were lower in cells treated with BioA than in the cell group treated with the known mitochondrial OXPHOS uncoupler FCCP. Next, we assessed mitochondrial membrane potential through JC-1 staining, which measures the formation of JC-1 aggregates (Figure 3F). High accumulation of JC-1 aggregates confirmed normal numbers of healthy and functional mitochondria in C3H10T1/2 cells treated with BioA. As shown in Figure 3F, BioA treatment did not adversely affect mitochondrial health compared to cells treated with MDI (positive control). However, FCCP treatment (negative control) decreased mitochondrial membrane potential, and co-treatment with FCCP and BioA did not reverse the inhibitory effect of FCCP treatment. These data suggest that BioA supplementation promotes mitochondrial biogenesis, resulting in high expression of UCP1.

2.4. BioA induces a brown fat phenotype in C3H10T1/2 MSCs

To determine the effect of BioA on thermogenesis, we first examined the expression of common thermogenic genes, including protein expression. BioA treatment significantly increased the mRNA expression of Ucp1, Pgc1α, Prdm16, and Pparγ genes (3.7-, 2.2-, 1.5-, and 5.8-fold, respectively) and their protein expression compared to cells treated with MDI ( Figures 4A and 4B ). BioA treatment significantly increased Pparγ expression compared to cells treated with MDI, and it is also notable that Pparγ expression was similar in both cells treated with BioA and Rosi (a PPARγ agonist). Previously, it was reported that BioA acts as a PPARγ activator (Wang et al., 2014). Based on the current and previous findings, we speculated that BioA promoted the brown fat phenotype in a Pparγ-dependent manner. Next, we assessed common adipogenic differentiation genes and their protein expression. As shown in Figures 4C and 4D, BioA enhanced the expression of aP2 and Cebpβ. Next, we measured the expression of beige- and brown-specific genes. BioA treatment increased the expression of markers CD137 (1.5-fold) and Fgf21 (2.2-fold) in beige and Cox2 (1.2-fold), Elovl3 (5.0-fold) and Zic1 (1.8-fold) in brown compared to MDI-treated cells, but not others. The beige-specific genes Tbx1 and Tmem26 were not significantly affected (Figure 4E). As expected, BioA reduced the expression of white fat-specific genes such as leptin, Asc1, Serpina3K, Psat1, and Tcf21 (Figure 4F). Additionally, BioA reduced the expression of myogenic and chondrogenic genes. These findings suggested that BioA could induce a brown fat phenotype.

2.5. BioA activates AMPK signaling during brown fat phenotype enhancement in C3H10T1/2 MSCs

To test whether BioA promotes the brown fat phenotype through AMPK signaling, the amounts of total AMPK protein and p-AMPK were measured at different time points: 0, 10, 30, and 60 min. As shown in Figure 5A, the amount of p-AMPK decreased over time from 10 to 60 min in MDI-treated cells, whereas MDI treatment supplemented with BioA reversed the downregulation of p-AMPK protein. Therefore, we chose the 60 min time point of BioA treatment to perform chemical inhibition experiments with DRS (10 μM), a known AMPK inhibitor, to determine whether BioA could attenuate the inhibitory effect of DRS. As illustrated in Figure 5B, the amount of p-AMPK was much lower in DRS treated cells. In contrast, BioA treatment significantly upregulated p-AMPK and attenuated DRS-induced downregulation of p-AMPK. Additionally, we observed induction of phosphorylation of AMPK and expression of downstream proteins after incubation with AMPK activators, AICAR and BioA. To confirm the activating effect of BioA on AMPK signaling and eliminate false positive results from chemical inhibition experiments, we performed siAmpkα-mediated gene knockdown and assessed the expression of Ampkα and its downstream genes, including protein levels. As shown in Figure 5C, siAmpkα transfection reduced the mRNA expression of the Ampkα (Prkaa1) gene by approximately 90%, but when siAmpkα transfected cells were incubated with BioA, the expression of these genes increased more than two-fold (approximately 2.8-fold). ; levels in siAmpkα-transfected cells) were restored. Nevertheless, it is noteworthy that treatment with BioA alone in differentiation cocktail (MDI) enhanced the expression of Ampkα by up to 22.7-fold. Next, we identified thermogenic genes downstream of Ampkα (Ucp1, Pgc1α, Prdm16, Pparγ and We confirmed the expression of Sirt1). As shown in Figures 5C and 5D, the mRNA and protein expression levels of Ucp1, Pgc1α, Prdm16, and Pparγ in the siAmpkα transfection group were much lower. Additionally, the expression of Sirt1, a mitochondrial biogenesis-related gene, was significantly lower. Expression was significantly lower in siAmpkα-transfected cells.In contrast, in the group of siAmpkα-transfected cells treated with BioA, expression of thermogenic markers was restored slightly in terms of mRNA but significantly in terms of protein amount. , Pparγ mRNA expression was significantly higher in siAmpkα-transfected cells treated with BioA than in cells treated with BioA alone. Additionally, expression levels of thermogenic markers were similar between MDI-treated and siCont-treated cells, suggesting that siAmpkα knockdown The experiment using was confirmed to be valid. In contrast, BioA treatment increased the expression of thermogenic markers compared to MDI-treated cells. These data suggest that BioA induces thermogenesis through the AMPK signaling pathway and AMPK activation. This provided evidence that AMPK activity can be promoted when inhibited by DRS or siAmpkα-based knockdown.

3. Considerations

Flavonoids are a class of diverse phytochemicals that have been widely studied for their involvement in regulating thermogenesis in adipose tissue (Rasbach & Schnellmann, 2008; Zhang et al., 2019). BioA, a soy isoflavone, is known to have various pharmacological properties, including weight loss, anti-diabetic effects, inhibition of lipid accumulation, osteogenic activity, and adipogenic differentiation, thereby alleviating obesity and obesity-related metabolic diseases (Harini, Ezhumalai, & Pugalendi, 2012; Oza & Kulkarni, 2018; Raheja, Girdhar, Lather, & Pandita, 2018). According to a clinical study, BioA administration was found to lower LDL cholesterol levels in humans (Nestel et al., 2004). In adipose tissue, BioA has anti-adipogenic activity in primary adipose-derived stem cells (Su et al., 2013), stimulation of adipogenesis in 3T3-L1 preadipocytes (Shen et al., 2006), and promotion of mitochondrial biogenesis (Rasbach & Schnellmann, 2008), and has various therapeutic effects. Although there is extensive available information on the impact of BioA on adipose tissue, the impact of BioA on the formation of brown fat-like adipocytes from MSCs has not yet been studied. Accordingly, here we demonstrated that BioA induces brown fat phenotype expression, with mitochondrial biogenesis and lipolysis being the major contributing factors in promoting brown fat phenotype in C3H10T1/2 MSCs.

C3H10T1/2 MSCs tend to differentiate into brown-like adipocytes when cultured with adipogenic induction medium containing Rosi, T3, and BMP7 in the early stages of adipogenic differentiation (Imran et al., 2017; Wang et al., 2018; Yoon , Imran, & Kim, 2018). In this study, we observed that treatment with BioA alone could induce brown-like adipocyte characteristics in C3H10T1/2 MSCs, as evidenced by upregulation of Ucp1, Pgc1α, Prdm16 and Pparγ. Previously, a fluorescent PPARγ ligand-binding assay and a chimeric Gal-PPAR reporter gene bioassay showed that both pioglitazone and BioA had maximal effective concentrations of 3.0 (approximately 40-fold increase in PPARγ activity) and 3.7 μmol/L, respectively. It has been shown to be a potent PPARγ activator with (EC ₅₀ ) (Shen et al., 2006). In the same study, they demonstrated that BioA enhances PPARγ activity by up to 7.6-fold, as judged by a PPAR-induced reporter gene (CYP4A6-PPRE-Luciferase) assay on three differentiated T3-L1 adipocytes ( Shen et al., 2006). In our study, we observed that treatment with BioA alone enhanced PPARγ expression by up to 4-fold, but when applied to siAmpkα-transfected (Ampkα knockdown) cells, its expression increased by up to 7.6-fold. The widely studied PPARγ agonist Rosi is known to induce the browning process in human adipose-derived MSCs (Loft et al., 2015), and in this study, we confirmed that, similar to Rosi, BioA increases PPARγ activity. Therefore, we speculated that BioA promotes the brown fat phenotype in a PPARγ-dependent manner. Moreover, BioA slightly upregulated PRDM16. It has been reported that PPARγ can induce browning of WAT by stabilizing the expression of PRDM16 (Ohno, Shinoda, Spiegelman, & Kajimura, 2012). Therefore, it can be assumed that BioA promotes the brown fat phenotype by activating PPARγ and/or PRDM16. PGC1α, a PPARγ coactivator, has been extensively studied due to its broad activity in several thermogenic processes, such as mitochondrial biogenesis, β-oxidation, and lipid metabolism (Cheng, Ku, & Lin, 2018). Our confirmation results and previous findings indicate that BioA increases PGC1α expression during the development of the brown fat phenotype (Rasbach & Schnellmann, 2008). Additionally, BioA supplementation upregulated key beige (Cd137 and Fgf21) and brown (Elovl3 and Zic1) specific markers and the early adipogenic differentiation marker CEBPβ. This means that BioA enables C3H10T1/2 MSCs to generate brown and partially beige-specific lineages in the early stages of differentiation.

The expression of brown fat-like phenotype is closely related to mitochondrial biogenesis. Over the past decade, research activities have focused on discovering ways to improve mitochondrial biogenesis through genetic manipulation or pharmacological intervention (Mueller, 2016; Villena et al., 2002). Isoflavones have been reported to improve thermogenesis by promoting mitochondrial biogenesis. BioA and its catabolic product, genistein, have been demonstrated to induce mitochondrial biogenesis through activation of PGC1α and SIRT1 (Rasbach & Schnellmann, 2008). PGC1α and SIRT1-dependent induction of mitochondrial biogenesis is regulated by AMPK signaling, resulting in high oxygen consumption and cellular ATP production (Artsi et al., 2019; Rasbach & Schnellmann, 2008). Additionally, we observed that BioA treatment significantly enhanced SIRT1 expression and slightly induced the expression of other mitochondrial-biogenesis markers COX8b, CIDEA and DIO2. Additionally, BioA upregulated oxygen consumption, which is known to be coupled to OXPHOS during ATP production. It is noteworthy that BioA administration increased mtDNA amount and FIS1 expression without affecting mitochondrial membrane potential. In contrast, BioA cannot reverse the inhibitory effect of the mitochondrial uncoupler FCCP, but it is noteworthy that it has no serious side effects when applied alone. From these findings, it is believed that BioA promotes thermogenesis without degrading functional mitochondria. Therefore, BioA can increase the amount of mtDNA through activated mitochondrial fission without disrupting functional mitochondria in C3H10T1/2 MSCs.

Induction of thermogenesis through improvement of mitochondrial biogenesis requires FFAs as a fuel source produced by lipolysis (Chouchani & Kajimura, 2019). Lipolysis generally occurs in WAT during the browning process (Bolsoni-Lopes & Alonso-Vale, 2015). In our previous study, we demonstrated that HSL and ATGL, two common lipolysis markers, are highly upregulated during lipolysis (Yoon et al., 2018). There has been debate as to whether both AMPK and PKA signaling cascades are involved in the phosphorylation of ATGL at S406 to induce lipolysis. Additionally, AMPK phosphorylation is known to result in HSL phosphorylation at S565, thereby inhibiting PKA-mediated phosphorylation at S563 and S660 (Ahmadian et al., 2011). In contrast, we found that BioA increased the expression of the genes Hsl, Atgl, Mgll, and Abhd5. For protein expression, BioA treatment upregulated ATGL and HSL phosphorylated at S660 and S563. Nevertheless, in AMPK-mediated lipolysis, HSL phosphorylation at S660 and S563 is generally suppressed (Kim et al., 2016; Larsson, Jones, Goransson, Degerman, & Holm, 2016). Morphological changes, such as alterations in lipid droplet size, by BioA treatment ensure lipolysis in C3H10T1/2 MSCs. Although it is clear that BioA induces lipolysis, further studies are needed to confirm its mechanism of action. In addition to lipolysis, BioA upregulated the mitochondrial β-oxidation gene Lcad while downregulating adipogenic genes and white adipocyte-specific genes. It is also worth mentioning that BioA treatment terminates differentiation into other cell types such as myoblasts, osteoblasts and chondrocytes and only induces brown fat-like adipocyte formation in C3H10T1/2 MSCs.

Several studies have shown that the AMPK signaling pathway regulates key thermogenic processes during browning of WAT, BAT activation, brown fat-like differentiation of MSCs, mitochondrial biogenesis, respiration, lipolysis, and β-oxidation of lipids. (Ahmadian et al., 2011; Imran et al., 2017; Rahman & Kim, 2020a; Zhang et al., 2019). In our study, we confirmed that BioA upregulates brown fat-like phenotypes by inducing mitochondrial biogenesis and respiration, lipolysis, lipid β-oxidation, and activation of AMPK signaling. Our gene knockdown and competitive chemical inhibition experiments showed that upregulation of thermogenesis-related genes and mitochondria-biogenesis-related genes was mediated by AMPK signaling in BioA-treated cells. AMPK has been reported to play a pivotal role in the differentiation and function of brown or beige adipocytes from mesenchymal stem cells (Abdul-Rahman et al., 2016; Imran et al., 2017; Imran, Yoon, & Kim, 2018; Jeong et al. , 2017; Nagy et al., 2019). Stimulation of AMPK signaling causes SIRT1-mediated deacetylation of PPARγ, producing brown fat-like features and consequently improving mitochondrial biogenesis. The activities of AMPK and SIRT1 are interconnected and regulated by small molecule enzyme cofactors such as NAD ⁺ (Canto et al., 2009). It is known that disruption of NAD ⁺ or AMPK expression results in loss of brown fat-like phenotype ( Abdul-Rahman et al., 2016 ; Canto et al., 2009 ; Nagy et al., 2019 ; Qiang et al., 2012 ). Therefore, considering previous studies, we proposed that BioA enhances the brown fat phenotype through activation of mitochondrial biogenesis and AMPK-SIRT1-PPARγ signaling in murine C3H10T1/2 MSCs.

However, we also investigated whether several other signaling pathways, such as PKA, Smad1/5 and p38 MAPK, may contribute to the formation of brown fat-like adipocytes. Our validation results suggested that BioA acts only through AMPK signaling and other signals may not be involved in brown-like adipogenesis in murine C3H10T1/2 MSCs.

4. Conclusion

This study provides comprehensive evidence that BioA induces brown fat phenotype upregulation in C3H10T1/2 MSCs through activation of AMPK signaling. Additionally, it indicates that BioA treatment promotes mitochondrial biogenesis and lipolysis, which regulates the thermogenic process. BioA improves energy expenditure by promoting mitochondrial respiration without disrupting the cell's functional mitochondria. These confirmatory results suggest that BioA may be a new anti-obesity drug but requires clinical studies.

<110> SOONCHUNHYANG INDUSTRY-ACADEMY COOPERATION FOUNDATION <120> A PHARMACEUTICAL COMPOSITION FOR PREVENTION OR TREATMENT OF OBESITY COMPRISING BIOCHANIN A OR PHARMACEUTICALLY ACCEPTED SALTS THEREOF AS AN EFFECTIVE COMPONENT <130> YP200028 <160> 88 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Ucp1 forward primer <400> 1 ggcattcaga ggcaaatcag ct 22 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Ucp1 reverse primer <400> 2 caatgaacac tgccacacct c 21 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Prdm16 forward primer <400> 3 cagcacggtg aagccattc 19 <210> 4 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Prdm16 reverse primer <400> 4 gcgtgcatcc gcttgtg 17 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Sox9 reverse primer <400> 5 acagctttct gggtggatt 19 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Pgc1a reverse primer <400> 6 tgaggaccgc tagcaagttt 20 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Pparg forward primer <400> 7 tttgaaagaa gcggtgaacc ac 22 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Pparg reverse primer <400> 8 accattgggt cagctcttgt g 21 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> aP2 forward primer <400> 9 gtgatgcctt tgtgggaaac ctggaag 27 <210 > 10 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> aP2 reverse primer <400> 10 tcataaactc ttgtggaagt cacgcc 26 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence < 220> <223> Psat1 forward primer <400> 11 taccgccttg tcaagaaacc 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Psat1 reverse primer <400> 12 agtggagcgc cagaatagaa 20 <210 > 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Leptin forward primer <400> 13 cacacacgca gtcggtatcc 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220 > <223> Leptin reverse primer <400> 14 agcccaggaa tgaagtccaa 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Asc1 forward primer <400> 15 gggtggcact caagaaagag 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Asc1 reverse primer <400> 16 agtgttccag gacacccttg 20 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Serpina3K forward primer <400> 17 ggctgaaggc aaagtcagtg t 21 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Serpina3K reverse primer <400> 18 tggaatctgt cctgctgtcc t 21 <210 > 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Tcf21 forward primer <400> 19 cattcaccca gtcaacctga 20 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220 > <223> Tcf21 reverse primer <400> 20 ttccttcagg tcattctctg g 21 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CD137 forward primer <400> 21 cgtgcagaac tcctgtgata ac 22 < 210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CD137 reverse primer <400> 22 gtccacctat gctggagaag g 21 <210> 23 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Tbx1 forward primer <400> 23 ggcaggcaga cgaatgttc 19 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Tbx1 reverse primer <400> 24 ttgtcatcta cgggcacaaa g 21 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Zic1 forward primer <400> 25 ctgttgtggg agacacgatg 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Zic1 reverse primer <400> 26 ctgttgtggg agacacgatg 20 <210> 27 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Cox2 forward primer <400> 27 gactgggcca tggagtgg 18 < 210> 28 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Cox2 reverse primer <400> 28 cacctctcca ccaatgacc 19 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence < 220> <223> Cox1 forward primer <400> 29 tctactattc ggagcctgag 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Cox1 reverse primer <400> 30 ctactgatgc tcctgcatgg 20 <210 > 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Adrb3 forward 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catctacatc a 21 < 210> 40 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Abhd5 reverse primer <400> 40 cagcgtccat attctgtttc ca 22 <210> 41 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Dgat2 forward primer <400> 41 ccgcaaaggc tttgtgaa 18 <210> 42 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Dgat2 reverse primer <400> 42 ggaataagtg ggaaccagat cag 23 <210> 43 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Acc forward primer <400> 43 tgaccgtggg cacaaagtt 19 <210> 44 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Acc reverse primer <400> 44 aggaggaacc gcatttatcg a 21 <210> 45 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LCAD forward primer <400> 45 aaggatttat taagggcaag aagc 24 <210> 46 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> LCAD reverse primer <400> 46 ggaagcggag gcggagtc 18 <210> 47 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Mfn1 forward primer <400> 47 tctccaagcc caacatcttc a 21 <210> 48 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Mfn1 reverse primer <400> 48 actccggctc cgaagca 17 <210> 49 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mfn2 forward primer <400> 49 acagcctcag ccgacagcat 20 <210> 50 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Mfn2 reverse primer <400> 50 tgccgaagga gcagacctt 19 <210> 51 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Opa1 forward primer <400> 51 cagctggcag aagatctcaa g 21 <210> 52 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Opa1 reverse primer <400> 52 tatgagcagg attttgacca c 21 <210> 53 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Drp1 forward primer <400> 53 ctgacgcttg tggatttacc 20 <210> 54 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Drp1 reverse primer <400> 54 cccttcccat caatacatcc 20 <210> 55 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Fis1 forward primer <400> 55 gcccctgcta ctggaccat 19 <210> 56 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Fis1 reverse primer <400> 56 ccctgaaagc ctcacactaa gg 22 <210> 57 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Sirt1 forward primer <400> 57 gcatagatac cgtctcttga tctgaa 26 <210> 58 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Sirt1 reverse primer <400> 58 tgtgaagtta ctgcaggagt gtaaa 25 <210> 59 <211> 23 <212> DNA <213 > Artificial Sequence <220> <223> Dio2 forward primer <400> 59 cagtgtggtg cacgtctcca atc 23 <210> 60 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Dio2 reverse primer <400> 60 tgaacccccg ttgaccacca g 21 <210> 61 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nrf1 forward primer <400> 61 caacagggaa gaaacggaaa 20 <210> 62 <211> 20 <212> DNA < 213> Artificial Sequence <220> <223> Nrf1 reverse primer <400> 62 gcaccacatt ctccaaaggt 20 <210> 63 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Cebpb forward primer <400> 63 cagctgctcc accttcttct 20 <210> 64 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Cebpb reverse primer <400> 64 caagctgagc gagtaca 17 <210> 65 <211> 20 <212> DNA <213 > Artificial Sequence <220> <223> Fgf21 forward primer <400> 65 agatcaggga ggatggaaca 20 <210> 66 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Fgf21 reverse primer <400> 66 tcaaagtgag gcgatccata 20 <210> 67 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> p0 forward primer <400> 67 gcactttcgc tttctggagg gtgt 24 <210> 68 <211> 24 <212> DNA <213 > Artificial Sequence <220> <223> p0 reverse primer <400> 68 tgacttggtt gctttggcgg gatt 24 <210> 69 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Cox7a forward primer <400> 69 cagcgtcatg gtcagtctgt 20 <210> 70 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Cox7a reverse primer <400> 70 agaaaaccgt gtggcagaga 20 <210> 71 <211> 20 <212> DNA <213 > Artificial Sequence <220> <223> Cox8b forward primer <400> 71 gaaccatgaa gccaacgact 20 <210> 72 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Cox8b reverse primer <400> 72 cgcaagttca cagtcgttcc 20 <210> 73 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Tmen26 forward primer <400> 73 accctgtcat cccacagag 19 <210> 74 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Tmem26 reverse primer <400> 74 tgtttggtgg agtcctaagg tc 22 <210> 75 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Elov13 forward primer <400> 75 tccgcgttct catgtaggtc t 21 <210> 76 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Elov13 reverse primer <400> 76 ggacctgatg caaccctatg a 21 <210> 77 <211> 21 <212> DNA < 213> Artificial Sequence <220> <223> Cidea forward primer <400> 77 tgctcttctg tatcgcccag t 21 <210> 78 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Cidea reverse primer <400> 78 gccgtgttaa ggaatctgct g 21 <210> 79 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Tbp forward primer <400> 79 gaagctgcgg tacaattcca g 21 <210> 80 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Tbp reverse primer <400> 80 ccccttgtac ccttcaccaa t 21 <210> 81 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> MyoD forward primer < 400> 81 cgccactccg ggacatag 18 <210> 82 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> MyoD reverse primer <400> 82 gaagtcgtct gctgtctcaa agg 23 <210> 83 <211> 24 <212 > DNA <213> Artificial Sequence <220> <223> MyoG forward primer <400> 83 agcgcaggct caagaaagtg aatg 24 <210> 84 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> MyoG reverse primer <400> 84 ctgtaggcgc tcaatgtact ggat 24 <210> 85 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Sox9 forward primer <400> 85 cggaggaagt cggtgaaga 19 <210> 86 <211> 19 < 212> DNA <213> Artificial Sequence <220> <223> Sox9 reverse primer <400> 86 gtcggttttg ggagtggtg 19 <210> 87 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Collal forward primer <400> 87 tggtgtccca catctacatc a 21 <210> 88 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Collal reverse primer<400> 88 cctcgggttt ccacgtctc 19

Claims (7)

delete delete delete delete A composition for inducing differentiation of mesenchymal stem cells into brown adipocytes in vitro, comprising Biochanin A or a pharmaceutically acceptable salt thereof as an active ingredient. The method of claim 5, wherein the induction of differentiation occurs through a mechanism selected from the group consisting of reducing intracellular triglyceride accumulation, inducing lipolysis, increasing the number of intracellular mitochondria, inducing a brown fat phenotype in cells, and activating AMPK signaling. A composition for inducing differentiation, characterized in that: A method of inducing differentiation into brown adipocytes comprising treating mesenchymal stem cells isolated from the human body with Biochanin A or a pharmaceutically acceptable salt thereof.