growth inhibitors of adipocytes

Abstract

[Problem] This invention provides a useful therapeutic tool for obesity-related lipogenesis using DHMBA. Cultures containing DHMBA inhibit the growth of 3T3-L1 precursor adipocytes in vitro, reducing their number. During adipocyte differentiation, if cultured in the presence of DHMBA, lipogenesis is inhibited through the regulation of various signaling processes related to insulin signaling, thus suppressing lipogenesis from 3T3-L1 precursor adipocytes. [Solution] The invention is characterized by its inhibitory effect on the growth of 3T3-L1 precursor adipocytes, with 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as the active ingredient.

Description

Growth inhibitor of adipocytes The present invention relates to a growth inhibitor of adipocytes. The enlargement of adipocytes in adipose tissue causes disordered pathophysiological states including type 2 diabetes, hyperlipidemia, hypertension, cardiovascular diseases, and cancer. The phenolic antioxidant 3,5-dihydroxy-4-methoxybenzyl alcohol (hereinafter also referred to as DHMBA) can act as an intracellular free radical scavenger to control oxidative stress. However, the role it plays in the improvement and repair of disease states has not been fully elucidated. The present invention investigated the regulatory effect of DHMBA on lipogenesis in mouse 3T3-L1 adipocytes in vitro. 3T3-L1 preadipocytes were cultured in DMEM medium containing 10% fetal bovine serum. When DHMBA was added to the culture medium, the proliferation of 3T3-L1 preadipocytes cultured in a culture medium without adipocyte differentiation factors was inhibited. Notably, in the case of culturing 3T3-L1 preadipocytes in a culture medium containing differentiation-inducing factors including insulin, DHMBA did not affect the cell number during the differentiation process of lipogenesis. When 3T3-L1 preadipocytes were cultured in a culture medium containing DHMBA (1, 10, or 100 μM), the lipid accumulation in adipocytes was inhibited, and even the lipogenesis of 3T3-L1 adipocytes was blocked. The strong inhibitory effect of DHMBA on lipogenesis was observed in the later stage of the culture with added insulin. Lipogenesis was blocked by the presence of the inhibitors Wortmannin, PD98059, or Bay 11-7082 of the pathway related to the insulin signaling pathway. In particular, the inhibitory effect of DHMBA on lipogenesis was exhibited even in the presence of these inhibitors. In addition, its mechanism of action is related to the decrease in the concentrations of PPARγ and C / EBPα related to the differentiation of preadipocytes and the concentrations of PI3 kinase 100α, Akt, MAPK, phosphorylated MAPK, and mTOR related to the insulin signaling pathway by DHMBA. [Prior art documents] [Patent documents] [Patent document 1] Japanese Patent Application Laid-Open No. 2017-132753 [Problems to be solved by the invention] In the present invention, DHMBA inhibits lipogenesis by regulating various signaling pathways, and can provide a new strategy for preventing and treating obesity. Generally speaking, obesity is caused by the excess energy generated from consuming high-calorie foods. In fat cells, triglycerides accumulate through fatty acids. The increase in fat cells in adipose tissue causes abnormal increases in the concentrations of hormones and cytokines secreted by this tissue, triggering symptoms of diseases such as type 2 diabetes, hyperlipidemia, hypertension, cardiovascular diseases, and cancer. Currently, obesity is recognized as a risk factor for various diseases. Therefore, adipose tissue plays an important role in maintaining pathophysiological stability. Adipose tissue is composed of fat cells formed by the differentiation of bone marrow mesenchymal stem cells into preadipocytes. Fat biosynthesis is an important process for the formation of fat cells and lipid accumulation. The main transcription factors that regulate this process are known to include CCAAT / enhancer-binding protein α (C / EBPα), peroxisome proliferator-activated receptor γ (PPARγ), sterol regulatory element-binding protein-1c (SREBP-1c), and fatty acid-binding protein (FABP4). C / EBPα and PPARγ are essential for the differentiation of precursor cells into mature fat cells. PPARγ is involved in promoting fat biosynthesis in cells lacking C / EBP expression. SREBP is a co-regulatory factor for fat biosynthesis and plays an important role in the regulation of lipid metabolism. These transcription factors regulate the synthesis of fatty acids and triglycerides in fat biosynthesis. In addition, it is also known that mitogen-activated protein kinase (MAPK) and protein kinase B (AKT), which are related to the insulin signaling pathway, activate fat biosynthesis in fat cells. The new phenolic antioxidant 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) was initially discovered in the Pacific oyster (Crassostrea Gigas). DHMBA has the two characteristics of capturing free radicals in certain cells and preventing oxidative stress. As an antioxidant, DHMBA may play an important nutritional role in the regulation of cell functions. Recently, it has been confirmed that DHMBA can inhibit the proliferation of metastatic prostate cancer cells by targeting various signaling pathways, and new strategies for treating prostate cancer using DHMBA have been proposed. In addition, it has been determined that the pharmacological effects of DHMBA are important for the prevention and treatment of various diseases. This invention was carried out to determine whether DHMBA regulates adipogenesis in 3T3-L1 preadipocytes in vitro. It was determined here that culturing with DHMBA inhibits the growth of 3T3-L1 preadipocytes in vitro, resulting in a decrease in cell number. Such an effect was not induced during the adipocyte differentiation process. It was found that during this cell differentiation process, when cultured in the presence of DHMBA, adipogenesis is inhibited through the regulation of various signal transduction programs related to insulin signal transduction during the differentiation of 3T3-L1 preadipocytes into adipocytes. This invention provides an effective therapeutic tool for adipogenesis related to obesity. [Means for Solving the Problem] This invention relates to: an inhibitor of adipocyte growth, characterized by having an inhibitory effect on the growth of 3T3-L1 preadipocytes with 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an active ingredient; or an inhibitor of lipid accumulation in adipocytes, characterized by having an inhibitory effect on lipid accumulation in adipocytes with 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an active ingredient; or an inhibitor of adipocyte generation, characterized by having an inhibitory effect on adipocyte generation with 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an active ingredient; or a regulator of the signal transduction pathway possessed by adipocytes, characterized by having a regulatory effect on the signal transduction pathway possessed by adipocytes with 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an active ingredient; or an accelerator for reducing the concentration of proteins related to the signal transduction program of adipogenesis in adipocytes, characterized by having a reducing effect on the concentration of proteins related to the signal transduction program of adipogenesis in adipocytes with 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an active ingredient. [Effects of the Invention] In the present invention, DHMBA inhibits lipogenesis by regulating various signal transduction pathways, and exhibits the effect of providing a new strategy for preventing and treating obesity. That is, it was determined that culturing with DHMBA inhibits the growth of 3T3-L1 preadipocytes, resulting in a decrease in cell number. In addition, it was also found that during the cell differentiation process, culturing in the presence of DHMBA inhibits lipogenesis during the differentiation of 3T3-L1 preadipocytes into adipocytes through the regulation of various signal transduction processes related to insulin signal transduction. Therefore, the present invention also exhibits the effect of providing an effective therapeutic tool for obesity-related lipogenesis using DHMBA. (Experimental Materials and Methods of the Present Invention) Reagents Dulbecco's Modified Eagle Medium (DMEM) containing 4.5 g / L glucose, L-glutamine, and sodium pyruvate, and antibiotics (100 μg / mL penicillin and 100 μg / mL streptomycin; P / S) were purchased from Corning (Mediatech, Inc., Manassas, Virginia, USA). Antibiotics were contained at 1% in DMEM. Fetal bovine serum (FBS) was purchased from Thermo Scientific HyClone (Logan, Utah, USA). Calf bovine serum (BCS), dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), Wortmannin, Bay 11-7082, and all other reagents were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). 3,5-Dihydroxy-4-methoxybenzyl alcohol (DHMBA) The new amphiphilic phenolic compound 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), which exhibits antioxidant properties, was isolated from the Pacific oyster (Crassostrea Gigas). Synthetic DHMBA was used in the present invention. The structure of DHMBA is shown in Figure 1. The purity of synthetic DHMBA was 100%. DHMBA was dissolved in 100% ethanol and stored at -20 °C until use. 3T3-L1 preadipocytes 3T3-L1 mouse embryonic fibroblasts (preadipocytes) were purchased from the American Type Culture Collection (Rockville, Maryland, USA). The cells were maintained in DMEM containing 10% BCS and 1% P / S at 37 °C and in 5% CO 2 and 95% air. The cells were cultured until 70 - 80% confluent and the culture medium was changed every other day. The cells were subcultured every 3 days. Measurement of cell proliferation and apoptosis To measure the progress of cell proliferation, 3T3-L1 preadipocytes (1 x 10 5 / ml) were cultured in medium (final concentration 1% ethanol) or culture medium containing DHMBA (10 μM) at 37 °C, 5% CO 2 and 95% air for 1, 2, 3, or 4 days. In addition, in the experiment for measuring cell proliferation, 3T3-L1 preadipocytes (1 x 10 5 / ml) were cultured in culture medium containing medium (final concentration 1% ethanol) or DHMBA (0.1, 1, 10, 100, or 1000 μM) for 3 days. In the experiment for measuring apoptosis, 3T3-L1 preadipocytes (1 x 10 5 / ml) were cultured in DMEM medium containing 10% BCS and 1% P / S for 3 days. The cells that had reached near confluence were further cultured for 48 hours [15, 18] in the presence of medium (final concentration 1% ethanol) or DHMBA (0.1, 1, 10, 100, or 1000 μM), and the change in the number of cells was investigated. After culturing, a sterile solution containing 0.05% trypsin and EDTA (0.1 ml per well) was added to PBS (Thermo Fisher Scientific, Waltham, Massachusetts, USA) without Ca 2+ / Mg 2+ and incubated for 2 minutes to detach the attached cells from each well. Then, 0.9 ml of DMEM containing 10% FBS was added to each well to prepare a cell suspension [15, 17, 18]. To measure the number of live cells, the culture medium containing suspended cells (0.1 ml) was mixed with 0.1 ml of 0.5% trypan blue staining solution. Live cells were counted using a cell counter (Line Seiki H-102P, Tokyo, Japan) and a hemocytometer (Sigma-Aldrich, St. Louis, Missouri, USA) under a microscope (Olympus MTV-3). In each measurement, the average of two counts was taken. The number of cells was expressed as the number per well of the culture dish. Measurement of differentiation and lipid accumulation of 3T3-L1 preadipocytes To determine the effect of DHMBA on the differentiation of 3T3-L1 preadipocytes, 3T3-L1 preadipocytes (5 x 10 4Cells / ml) were cultured in 10% bovine calf serum (BCS) for 3 days until near confluence. After reaching near confluence (day 0), the culture medium was changed to a differentiation induction medium of DMEM containing 10% FBS, 1% P / S, 0.5 mM IBMX, 1 μM dexamethasone, and insulin (10 μg / ml), and the cells were further cultured for 2 days. After 2 days of culture, to enhance lipogenesis, the medium was replaced with fresh differentiation medium of DMEM containing 10% FBS and insulin (10 μg / ml), and the cells were further cultured for 2 days. Initially, to quantify the effect of DHMBA on cell number, at day 0 or 2 days after the start of differentiation culture, vehicle (final concentration of 1% ethanol) or DHMBA (1 or 10 μM) was added to the differentiation induction medium. After 0 - 2 days or 2 - 4 days of differentiation culture, as described in the previous section, the number of cells attached to the culture dish was measured. To determine the effect of DHMBA on the lipid content of 3T3-L1 adipocytes, in a further experiment, from day 0 to 4 days after differentiation induction culture, vehicle (final concentration of 1% ethanol) or DHMBA (0.1, 1, 10, or 100 μM) was added to the differentiation induction medium for culture. To quantify the lipid content (triglyceride) after cell culture, the cells in the 24-well plate were gently washed twice with PBS and fixed with 4% formaldehyde dissolved in PBS for 30 minutes. Next, the fixed cells were washed three times with PBS and stained with Oil Red O solution (a solution of 1.5% Oil Red O dissolved in 100% isopropanol) at room temperature for 60 minutes

[16] . After staining, the staining solution was removed and the cells were washed with PBS. Images of the stained lipid (triglyceride) droplets were observed and photographed with a microscope (40× magnification) (Olympus IX71; Olympus Corporation, Tokyo, Japan). In addition, to quantify the lipid content, the Oil Red staining was extracted with isopropanol. 100% isopropanol (300 μl) was added to each well and shaken for 30 minutes to elute. Using the eluate, the absorbance was measured at 510 nm with a spectrophotometer (μQuant, Bio-Tek Instruments). Measurement of Lipogenesis in 3T3-L1 Adipocytes 3T3-L1 preadipocytes (10 per well of a 96-well plate) 4Cells (0.2 ml) were cultured in DMEM containing 10% BCS and 1% P / S for 3 days until near confluence. After near confluence (day 0), cells were cultured in a differentiation induction medium containing 10% FBS, 1% P / S, 0.5 mM IBMX, and 1 μM dexamethasone with either vehicle (final concentration of 1% ethanol) or DHMBA (0.1, 1, 10, or 100 μM). After 2 days of culture, the medium was replaced with DMEM without 10% FBS and DHMBA but containing insulin (10 μg / ml), and the cells were further cultured for 2 days. In another experiment, cells were cultured in a differentiation medium without DHMBA on day 0 of differentiation induction. After 2 days of culture, the medium was replaced with either vehicle (final concentration of 1% ethanol) or DHMBA (0.1, 1, 10, or 100 μM) and DMEM containing 10% FBS and insulin (10 μg / ml), and the cells were further cultured for 2 days. Further in other experiments, to explore the association of intracellular signaling factors, after 2 days of differentiation culture, the medium was replaced with DMEM containing 10% FBS and insulin (10 μg / ml) and a medium containing either vehicle (1% ethanol) or DHMBA (1 or 10 μM). Cells were further cultured for 2 days in a medium containing signaling inhibitors such as genistein (1 or 10 μM), Wortmannin (10 or 100 nM), PD98059 (1 or 10 μM), or Bay 11-7082 (1 or 10 nM). After cell culture, the amount of intracellular lipid (triglyceride) formation was quantified using the AdipoRed assay reagent (Lonza, Walkersville, MD, USA) [19 - 21]. Fluorescence was measured using a 96-well plate reader (μQuant, Bio-Tek Instruments) with excitation at 485 nm and emission at 570 nm. Western blotting 3T3-L1 preadipocytes (using 1x10 6Cells (in a 100 mm culture dish, 10 ml of culture medium), cultured in DMEM containing 10% BCS and 1% P / S for 3 days until nearly confluent. Then, the cells were cultured for 2 days in a DMEM differentiation medium containing 10% FBS, 1% P / S, 0.5 mM IBMX, and 1 μM dexamethasone. Then, the culture medium was changed to a DMEM culture medium containing 10% FBS and insulin (10 μg / ml), with or without DHMBA (10 μM), and the cells were further cultured for 2 days. After culturing, the culture dish was washed 3 times with cold PBS (10 ml) to remove floating cells and dead cells, and the attached cells were scraped off using a cell lysate containing protease and protein phosphatase inhibitors (Roche Diagnostics, Indianapolis, Indiana, USA). Next, the cell lysate was centrifuged at 17,000 x g for 10 minutes at 4°C. The protein concentration in the supernatant was quantified using Bio-Rad Protein Assay Dye (Bio-Rad Laboratories Inc., Hercules, California, USA), with bovine serum albumin as the standard. The extracted protein samples were stored at -80°C until use. As disclosed in a previous invention, 40 μg of the supernatant protein sample per lane was separated by SDS polyacrylamide gel electrophoresis (12% SDS-PAGE) and transferred to a PVDF membrane. Specific antibodies against target proteins such as PPARγ (C26H12) (catalog number 2435, rabbit antibody), C / EBPα (catalog number 2295, rabbit), phosphoinositide 3-kinase p110α (PI3K, catalog number 4255), Akt (catalog number 9272, rabbit antibody), mitogen-activated protein kinase (MAPK, catalog number 4695, rabbit antibody), phosphorylated-MAPK (catalog number 4695, rabbit antibody), mammalian target of rapamycin (mTOR, catalog number 4517, mouse antibody), NF-κB p65 (catalog number 3034, rabbit antibody), β-actin (catalog number 3700, rabbit antibody), etc. were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA). For immunoblotting of the PVDF membrane, the above protein antibodies were used and incubated overnight at 4°C. After incubation, the PVDF membrane was incubated in a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology Inc., mouse sc-2005 or rabbit sc-2305, diluted 1:1,000) for 60 minutes at room temperature. The protein electrophoresis bands transferred to the PVDF membrane were detected on X-ray film using a chemiluminescent substrate (catalog number 34577, Thermo Scientific, Rockford, Illinois, USA).Data were from four independent experiments. The film was scanned using an Epson Perfection 1660 optical scanner, and the electrophoretic bands were quantified using Image J2 software (National Institutes of Health, Bethesda, MD, USA). Statistical analysis Statistical significance was determined using GraphPad InStat ver. 3 for Windows XP (GraphPad Software Inc., La Jolla, CA, USA). Data are presented as mean ± standard deviation (SD). Multiple comparisons were performed by one-way analysis of variance (ANOVA) with Tukey-Kramer multiple comparison test after parametric data testing. A p-value of < 0.05 was considered statistically significant. (Experimental results) (Effect of DHMBA on the proliferation of 3T3-L1 preadipocytes) First, we investigated whether DHMBA affects the growth of 3T3-L1 preadipocytes (Figure 2). Figure 2 is an illustration showing the effect of the marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) on cell proliferation or apoptosis in 3T3-L1 mouse embryonic fibroblasts (preadipocytes). Panel A of Figure 2 shows cells (1 x 10 5 cells / ml) cultured in DMEM medium containing 10% bovine calf serum (BCS) and 1% P / S in the presence of DHMBA (10 μM) for 1, 2, 3, 4, or 5 days. Panel B of Figure 2 shows cells (1 x 10 5 cells / ml) cultured for 3 days in medium (final concentration of 1% ethanol) or in the presence of DHMBA (final concentrations of 0.1, 1, 10, 100, or 1000 μM) to investigate the effect of increasing DHMBA concentration on cell proliferation. Panel C of Figure 2 shows cells (1 x 10 5 cells / ml) cultured for 3 days until near confluence, then DHMBA (final concentrations of 0.1, 1, 10, 100, or 1000 μM) was added and the cells were cultured for an additional 48 hours to investigate the effect of DHMBA on apoptosis. After culture, the number of cells attached to the culture dish was counted. Data were obtained from 8 wells of a cell culture plate using different cell preparations and are presented as mean ± SD (standard deviation). In addition, compared with the control group without DHMBA (gray bars), p < 0.001, indicating a significant difference. The significant difference test was based on one-way ANOVA and Tukey-Kramer post hoc test. Cells (10 cells / ml in each well of a 24-well plate) were cultured for 1, 2, 3, 4, and 5 days in the presence of medium (final concentration of 1% ethanol) or DHMBA (10 μM). The proliferation of cells was inhibited by culturing with DHMBA for 3 - 5 days. Therefore, it was determined that DHMBA inhibits the growth of 3T3-L1 preadipocytes in vitro (Figure 2A). Next, the cells were cultured for 3 days in the presence of medium or DHMBA (0.1, 1, 10, 100, or 1000 μM) with increased concentration. The proliferation of cells was inhibited at concentrations in the range of 1 - 1000 μM DHMBA (Figure 2B). In addition, to investigate the effect of DHMBA on apoptosis of 3T3-L1 preadipocytes, the cells were cultured in DMEM containing 10% FBS and 1% P / S for 3 days until nearly confluent, and then DHMBA (0.1, 1, 10, 100, or 1000 μM) was added to the culture medium containing 10% FBS and 1% P / S, and the cells were further cultured for 2 days (Figure 2C). The number of cells decreased due to the presence of DHMBA (1000 μM). This indicates that the higher the concentration of DHMBA, the more apoptosis is induced. 5 (Effect of DHMBA on cell proliferation during the differentiation process of 3T3-L1 preadipocytes) Next, the effect of DHMBA on cell proliferation during the differentiation process of 3T3-L1 preadipocytes was investigated (Figure 3). Figure 3 is an explanatory diagram showing the effect of the marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) on the number of cells during the cell differentiation process of 3T3-L1 mouse preadipocytes. Panel A of Figure 3 shows the cell culture process. Panel B of Figure 3 shows the cells (1x10 cells / ml in each well of a 24-well plate) cultured in DMEM containing 10% BCS and 1% P / S for 3 days, and then replaced with DMEM culture medium containing cell differentiation inducer 0.5 mM IBMX, 1 μM dexamethasone, and insulin (10 μg / ml), and medium (final concentration of 1% ethanol) or DHMBA (1 or 10 μM) was added and cultured for 2 days. Panel C of Figure 3 shows that after further culturing for 2 days (2 days after the differentiation induction culture), the culture medium was replaced with a medium containing only insulin (100 μg / ml) without IBMX and dexamethasone, and DHMBA (final concentration 1 or 10 μM) was added and the cells were further cultured for 2 days. 5 ​​After each culture, the number of cells attached to the culture dish was counted. The data were measured from 8 wells of a total of two duplicate culture plates using different cell preparations and are expressed as the mean ± standard deviation. The significant difference test was based on one-way ANOVA and Tukey-Kramer post hoc test. Cells that were nearly confluent after 3 days of culture (day 0) were further cultured for 2 days in DMEM culture solution containing a differentiation inducer of 10% FBS, 1% P / S, 0.5 mM IBMX, 1 μM dexamethasone, and insulin (10 μg / ml) with the addition of DHMBA (1 or 10 μM) (A in Figure 3). The number of cells did not change due to the presence of DHMBA (B in Figure 3). In addition, 2 days after adipogenesis was enhanced, the cells were further cultured for 2 days in DMEM culture solution containing insulin (10 μg / ml) with (1 or 10 μM) or without DHMBA. The number of cells did not change due to the presence of DHMBA (C in Figure 3). From these results, it was clarified that even when DHMBA was added during the differentiation process of adipogenesis, it had no effect on the number of cells. (DHMBA inhibits lipid accumulation in 3T3-L1 adipocytes) Figure 4 is an explanatory diagram showing the effect of the marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) on the production of lipid accumulation in 3T3-L1 mouse preadipocytes. 3T3-L1 preadipocytes (5 x 10 per well of a 24-well plate) 4Cells / ml) were cultured in DMEM medium containing 10% BCS and 1% P / S for 3 days. Using these cells that had reached near confluence, on day 0 when cell differentiation began, the medium was replaced with a differentiation factor-containing medium containing 10% fetal bovine serum (FBS), 1% P / S, 0.5 mM IBMX, 1 μM dexamethasone, and insulin (10 μg / ml), and the cells were further cultured for 2 days. Two days after the induction of cell differentiation culture, in order to further enhance lipogenesis, the medium was replaced with a fresh 10% FBS and insulin (10 μg / ml)-containing medium (DMEM) with lipogenesis-promoting factors, and the cells were further cultured for 2 days. To quantify the effect of DHMBA on lipogenesis in 3T3-L1 cells, on day 0 and 2 days after differentiation culture, the differentiation medium was supplemented with vehicle (final concentration 1% ethanol) or DHMBA (final concentrations 0.1, 1, 10, or 100 μM). After cell culture, the cells attached to the 24-well plate were gently washed twice with PBS and fixed with 4% formaldehyde dissolved in PBS for 30 minutes. Then, the fixed cells were washed three times with PBS, and Oil Red O solution (1.5% Oil Red O in isopropanol) was added and stained for 60 minutes. Panel A of Figure 4 is an explanatory diagram of the image of the stained lipid-accumulating cells observed under a microscope and photographed after staining. Panel B of Figure 4 is an explanatory diagram for quantifying the lipid (triglyceride) content. After Oil Red staining, the cells were extracted with isopropanol, and the absorbance of each well was measured at 510 nm using a spectrophotometer (μQuant, Bio-Tek Instruments). The data were obtained from 8 wells of two replicate culture plates using different cell preparations and are presented as mean ± standard deviation. In addition, compared with the control group without DHMBA (gray bars), p < 0.001, indicating a significant difference. The significant difference test was based on one-way ANOVA and Tukey-Kramer post hoc test. To determine the effect of DHMBA on lipid accumulation in 3T3-L1 adipocytes, on day 0 and 2 days after cell culture with a differentiation-inducing factor-containing medium, a vehicle medium (final concentration 1% ethanol) or DHMBA (0.1, 1, 10, or 100 μM) was added and cultured. After this culture, the fat (triglyceride) accumulated in the cells was stained with Oil Red O, and it was found that lipid accumulation in 3T3-L1 adipocytes decreased when cultured with DHMBA (Panel A of Figure 4). In addition, when measuring the intracellular lipid content, a decrease was confirmed (Panel B of Figure 4). These results indicate that DHMBA inhibits lipid accumulation in adipocytes. (DHMBA inhibits adipogenesis in 3T3-L1 adipocytes) Investigate whether adipogenesis in 3T3-L1 adipocytes changes due to culture with DHMBA (Figure 5). Figure 5 is an explanatory diagram showing the effect of the marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) on adipogenesis as 3T3-L1 mouse preadipocytes differentiate. The 3T3-L1 preadipocytes (1 x 10 per well of a 96-well plate) 4(cells / ml), cultured in DMEM medium containing 10% BCS and 1% P / S for 3 days until nearly confluent. Panel A of Figure 5 shows that after nearly confluence (day 0 of differentiation culture), the medium was replaced with DMEM medium containing differentiation-inducing factors including 10% fetal bovine serum (FBS), 1% P / S, 0.5 mM IBMX, 1 μM dexamethasone, and insulin, and vehicle (final concentration 1% ethanol) or DHMBA (final concentrations 0.1, 1, 10, or 100 μM) was added. After 2 days of culture, the medium was changed to fresh medium containing 10% FBS and insulin (10 μg / ml) (DMEM), and vehicle (final concentration 1% ethanol) or DHMBA (final concentrations 0.1, 1, 10, or 100 μM) was added, and the cells were further cultured for 2 days. Panel B of Figure 5 shows that after nearly confluence (day 0 of differentiation culture), the medium was replaced with a medium containing 10% fetal bovine serum (FBS), 1% P / S, 0.5 mM IBMX, 1 μM dexamethasone, insulin (10 μg / ml), and DHMBA (0.1, 1, 10, or 100 μM), and the cells were further cultured for 2 days. After 2 days of differentiation-inducing culture, the medium was changed to fresh medium containing 10% FBS and insulin (10 μg / ml) (without DHMBA), and the cells were further cultured for 2 days. Panel C of Figure 5 shows that after nearly confluence (day 0 of differentiation culture), the cells were cultured in a medium containing differentiation-inducing factors including 10% fetal bovine serum (FBS), 1% P / S, 0.5 mM IBMX, 1 μM dexamethasone, and insulin (10 μg / ml) for 2 days. Additionally, after 2 days of differentiation-inducing culture, the medium was changed to fresh medium containing 10% FBS, insulin (10 μg / ml), and DHMBA (0.1, 1, 10, or 100 μM), and the cells were further cultured for 2 days. After cell culture, commercially available AdipoRed assay reagent (Lonza, Walkersville, Maryland, USA) was used to measure the amount of lipid formation in the cells. Fluorescence was measured with a 96-well plate reader using excitation light at 485 nm and emission light at 570 nm. Data were obtained from 8 wells in a total of two replicate culture plates using different cell preparations and are expressed as mean ± standard deviation. Additionally, compared with the control group without DHMBA (gray bars), p < 0.001, indicating a significant difference. The significance test was based on one-way ANOVA and Tukey-Kramer post hoc test. Cells were cultured in DMEM containing 10% BCS and 1% P / S for 3 days. At the time point when the cells were nearly confluent, the cells were cultured in the presence of DHMBA (0.1, 1, 10, or 100 μM) for 4 days (days 0 to 4 after differentiation) (A in Fig. 5). When cultured with DHMBA (1, 10, or 100 μM), lipogenesis in 3T3-L1 adipocytes was inhibited (A in Fig. 5). Such inhibition could be observed in the culture after 0 to 2 days or 2 to 4 days after differentiation in the presence of DHMBA (1, 10, or 100 μM) (B and C in Fig. 5). In particular, the inhibition of lipogenesis in 3T3-L1 adipocytes cultured in the medium containing DHMBA was significant in the late stage of differentiation (2 to 4 days after) (C in Fig. 5). These results indicate that DHMBA strongly controls the program of lipogenesis that promotes insulin signaling in 3T3-L1 and adipocytes. (Effect of DHMBA on lipogenesis in 3T3-L1 adipocytes cultured with inhibitors of intracellular signaling pathways) To understand the mechanism by which DHMBA inhibits lipogenesis in 3T3-L1 adipocytes, the association of intracellular signaling factors was investigated (Fig. 6). Fig. 6 is an explanatory diagram showing the effect of the marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) on lipogenesis in 3T3-L1 mouse preadipocytes in the presence of various inhibitors of the signaling pathway. 3T3-L1 preadipocytes (1x10 per well of a 96-well plate) 4Cells / ml) were cultured in DMEM medium containing 10% BCS and 1% P / S for 3 days until nearly confluent. At the start of cell differentiation culture after nearly confluence (day 0 of differentiation induction culture), the medium was changed to DMEM medium containing 10% FBS, 1% P / S, 0.5 mM IBMX, 1 μM dexamethasone, and insulin (10 μg / ml), and the cells were cultured for an additional 2 days. Two days after this differentiation induction culture, the medium was replaced with fresh medium containing genistein (1 or 10 μM) (A in Figure 6), Wortmannin (10 or 100 nM) (B in Figure 6), PD98059 (1 or 10 μM) (C in Figure 6), or Bay 11-7082 (1 or 10 nM) (D in Figure 6), and vehicle (1% ethanol at the final concentration) or DHMBA (1 or 10 μM at the final concentration) was added, and the cells were cultured for an additional 2 days. After cell culture, the amount of intracellular lipid triglyceride formation was measured using the AdipoRed assay reagent (Lonza, Walkersville, Maryland, USA). Fluorescence was measured with a 96-well plate reader at an excitation wavelength of 485 nm and an emission wavelength of 570 nm. Data were obtained from 8 wells of a total of two replicate culture plates using different cell preparations and are expressed as the mean ± standard deviation. In addition, compared with the control group without DHMBA (gray bars), p < 0.001, indicating a significant difference. The significance test was based on one-way ANOVA and Tukey-Kramer post hoc test. Two days after cell differentiation, genistein (1 or 10 μM), Wortmannin (10 or 100 nM), PD98059 (1 or 10 μM), or Bay 11-7082 (1 or 10 nM) was added and the cells were cultured for 2 days. Lipid biosynthesis did not change in the presence of genistein (A in Figure 6), however, in the presence of genistein, DHMBA (1 or 10 μM) inhibited lipogenesis (A in Figure 6). In particular, lipogenesis was inhibited by the culture with Wortmannin (10 or 100 nM) (B in Figure 6), which is related to insulin signaling, PD98059 (1 or 10 μM) (C in Figure 6), or the NF-κB signaling inhibitor Bay 11-7082 (1 or 10 nM) (D in Figure 6). These inhibitions were further enhanced by the presence of DHMBA (1 or 10 μM), indicating that DHMBA inhibits lipogenesis by blocking various pathways including insulin and NF-κB signaling. (Effect of DHMBA on the protein concentrations related to the signal transduction process of lipogenesis) In addition, to understand the mechanism of action of the effect of DHMBA in inhibiting adipogenesis in adipocytes, the concentrations of important proteins involved in lipogenesis were investigated to see if they were regulated by DHMBA (A and B in Fig. 7). Fig. 7 is an explanatory diagram showing the effect of the marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) on the concentrations of various proteins related to the signal transduction of lipogenesis in adipocytes. The 3T3-L1 preadipocytes (1x10 6 cells / 100 mm culture dish, 10 ml culture medium) were cultured in DMEM medium containing 10% BCS and 1% P / S for 3 days until nearly confluent. At the start of induction of cell differentiation (day 0 of differentiation induction), the medium was changed to DMEM medium containing 10% FBS, 1% P / S, 0.5 mM IBMX, and 1 μM dexamethasone, and the cells were cultured for an additional 2 days. After these 2 days, the medium was changed to DMEM medium containing 10% FBS and insulin (10 μg / ml), DHMBA (final concentration 10 μM), and the cells were cultured for a further 2 days. After culturing, cell lysates were prepared. Protein samples from the centrifuged supernatant were used at 40 μg of protein per lane for Western blotting analysis. A in Fig. 7 shows representative data. The bars in B of Fig. 7 are expressed as the fold of the control group (without DHMBA). The data were measured from a total of 4 membranes using different cell preparations and are expressed as the mean ± standard deviation. In addition, compared with the control group, p < 0.01, indicating a significant difference. The significance test was based on one-way ANOVA and Tukey-Kramer post hoc test. When cells were cultured with the addition of DHMBA (10 μM), the concentrations of the transcription factors PPARγ and C / EBPα related to the differentiation of preadipocytes decreased. In addition, DHMBA also caused a decrease in the concentrations of PI3 kinase 100α, Akt, MAPK, phosphorylated MAPK, and mTOR related to the insulin signal transduction pathway during lipid biosynthesis. Furthermore, DHMBA also decreased the concentration of the transcription factor NF-κB p65. Therefore, it was found that when cultured with DHMBA, the concentrations of various protein molecules related to lipogenesis decreased. (Investigation) Adipose tissue is composed of adipocytes formed by the differentiation of bone marrow mesenchymal stem cells into preadipocytes. Adipose tissue plays an important role in maintaining the pathophysiological stability of type 2 diabetes, hyperlipidemia, hypertension, cardiovascular diseases, cancer, etc. DHMBA has been proven to capture free radicals in cells and has an antioxidant stress effect. In addition, in the present invention, the effect of DHMBA on adipogenesis has been studied. It has been found in the present invention that when 3T3-L1 preadipocytes are cultured in a culture solution containing DHMBA, the growth of the cells is inhibited. In addition, it has also been determined that DHMBA controls adipogenesis during the differentiation process of preadipocytes regardless of the manifestation of the inhibitory effect on cell growth in vitro. The present invention presents a new view that DHMBA has the effects of inhibiting adipocyte growth and inhibiting adipogenesis. Culturing adipocytes in the presence of DHMBA hinders the promotion of adipogenesis related to insulin signaling in 3T3-L1 and adipocytes. The adipogenesis inhibitory effect of this DHMBA is more strongly manifested in the later stage of the differentiation process compared to the initial stage of the differentiation process of 3T3-L1 preadipocytes. The initial stage of culturing is carried out in a culture solution containing the differentiation inducer IBMX, dexamethasone, and insulin, while the later stage of culturing contains only insulin. In particular, DHMBA strongly hinders adipogenesis in the later stage. Insulin exerts a strong promoting effect on adipogenesis and lipid accumulation in mature adipocytes. Therefore, it is considered that DHMBA strongly controls the program by which insulin enhances adipogenesis. The signaling pathway mechanism of obesity is very complex. In order to clarify the mechanism by which adipocyte culture in the presence of DHMBA inhibits adipogenesis, adipocytes were cultured in the presence of the tyrosine kinase inhibitor genistein related to the insulin signaling pathway, the PI3K / Akt signaling pathway inhibitor Wortmannin, the ERK / MAP kinase inhibitor PD98059, and the NF-κB inhibitor Bay 11-7089. As a result, even in the presence of these inhibitors, DHMBA still exerts an adipogenesis inhibitory effect. This result shows that DHMBA strongly hinders the adipogenesis of 3T3-L1 adipocytes by inhibiting various signaling programs related to insulin signaling. In addition, experiments were also conducted to clarify the mechanism by which DHMBA inhibits adipogenesis in adipocytes. It was found that culturing adipocytes with the addition of DHMBA decreased the concentrations of the transcription factors PPARγ and C / EBPα, which are related to the differentiation program of preadipocytes. These results support the view that DHMBA inhibits the process of differentiation from preadipocytes to adipocytes. In addition, culturing adipocytes with the addition of DHMBA also decreased the concentrations of the molecules PI3K, Akt, MAPK, phosphor-MAPK, and mTOR, which are involved in the promotion process of lipogenesis related to insulin signaling. The activation of the mTOR pathway promotes the differentiation of 3T3-L1 preadipocytes. These results further support the view that DHMBA regulates the lipogenesis program enhanced by insulin. In addition, DHMBA also decreased the concentration of NF-κB p65 in adipocytes. Lipogenesis is inhibited by the presence of the NF-κB signaling inhibitor Bay 11-7089. In addition, culturing with DHMBA inhibits lipogenesis in the presence of Bay 11-7089. These views illustrate that culturing adipocytes with the addition of DHMBA also inhibits the lipogenesis process regulated by the NF-κB signaling pathway. Figure 8 is an explanatory diagram showing the mechanism by which 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) inhibits lipogenesis in 3T3-L1 adipocytes. DHMBA decreases the concentrations of the transcription factors PPARγ and C / EBPα, which are related to the differentiation process of preadipocytes. In addition, DHMBA also decreases the concentrations of PI3K, Akt, MAPK, phosphorylated-MAPK, and mTOR, which are involved in the process of promoting lipogenesis related to insulin signaling. It is also illustrated that DHMBA also inhibits lipogenesis regulated by the NF-κB signaling pathway. In this way, DHMBA regulates various signaling pathways in adipocytes and exhibits an anti-lipogenic effect. As summarized in Figure 8, after investigation, it is considered that DHMBA exerts an anti-lipogenic effect by regulating various signaling pathways in adipocytes. In conclusion, it is known that DHMBA acts as a radical scavenger in cells and exhibits antioxidant stress, but in the present invention, it was further found that a new insight is that it exerts an effect of inhibiting the promotion of lipogenesis related to insulin signaling in mouse 3T3-L1 adipocytes in vitro. After investigation, it is considered that the effect of DHMBA is manifested through various pathways related to insulin signaling. Supplementation with DHMBA may have the potential to achieve the prevention and treatment of obesity by inhibiting adipogenesis in adipocytes. [Figure 1] shows the chemical structure of 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA). The molecular formula of DHMBA is C 8 H 10 O 4 , and its molecular weight is 170.164. [Figure 2] is an explanatory diagram showing the effect of the marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) on cell proliferation or apoptosis of 3T3-L1 mouse embryonic fibroblasts (preadipocytes). Panel A of Figure 2 shows the cells (1x10 5 cells / ml per well of a 24-well plate) cultured in DMEM medium containing 10% bovine calf serum (BCS) and 1% P / S in the presence of DHMBA (10 μM) for 1, 2, 3, 4, or 5 days. Panel B of Figure 2 is an explanatory diagram for investigating the effect of DHMBA on cell proliferation with increasing concentration. The cells (1x10 5 cells / ml per well of a 24-well plate) were cultured for 3 days in the medium (final concentration of 1% ethanol) or in the presence of DHMBA (final concentrations of 0.1, 1, 10, 100, or 1000 μM). Panel C of Figure 2 is an explanatory diagram for investigating the effect of DHMBA on apoptosis. The cells (1x10 5 cells / ml per well of a 24-well plate) were cultured for 3 days until they reached near confluence (subconfluent, 70% - 80% confluence), and then DHMBA (final concentrations of 0.1, 1, 10, 100, or 1000 μM) was added for an additional 48 hours of culture. After culture, the number of cells attached to the culture dish was counted. The data were measured from 8 wells of the cell culture plate using different cell preparations and are presented as the mean ± standard deviation (SD). In addition, compared with the control group without DHMBA (gray bars), p < 0.001, indicating a significant difference. The significance test was based on one-way ANOVA and Tukey-Kramer post hoc test. [Figure 3] is an explanatory diagram showing the effect of the marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) on the cell number during the cell differentiation process of 3T3-L1 mouse preadipocytes. Panel A of Figure 3 shows the cell culture process. Panel B of Figure 3 shows the cells (1x10 5After culturing for 3 days in DMEM containing 10% BCS and 1% P / S at (number of cells / ml), the medium was changed to DMEM containing 0.5 mM IBMX, 1 μM dexamethasone, and insulin (10 μg / ml) as cell differentiation inducing factors, and the cells were cultured for 2 days with the addition of vehicle (final concentration: 1% ethanol) or DHMBA (1 or 10 μM). Panel C of Fig. 3 shows the illustration of culturing for an additional 2 days (2 days after the differentiation induction culture), replacing the medium with DMEM containing only insulin (100 μg / ml) without IBMX and dexamethasone, and adding DHMBA (final concentration 1 or 10 μM), and further culturing the cells for 2 days. After each culture, the number of cells attached to the culture dish was counted. The data were obtained from 8 wells of two replicate culture plates using different cell preparations, and are expressed as mean ± standard deviation. The significance test was performed by one-way ANOVA and Tukey-Kramer post hoc test. [Fig. 4] is an illustration showing the effect of the marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) on lipid accumulation in 3T3-L1 murine preadipocytes. The 3T3-L1 preadipocytes (5x10 per well in a 24-well plate) 4(Cells / ml) were cultured in DMEM medium containing 10% BCS and 1% P / S for 3 days. Using the cells that had reached near confluence, on day 0 when cell differentiation began, the medium was replaced with a differentiation factor-containing medium containing 10% fetal bovine serum (FBS), 1% P / S, 0.5 mM IBMX, 1 μM dexamethasone, and insulin (10 μg / ml), and further cultured for 2 days. Two days after the induction of cell differentiation culture, to further enhance lipogenesis, the medium was replaced with a fresh medium containing 10% FBS and insulin (10 μg / ml) with lipogenesis-promoting factors (DMEM), and the cells were further cultured for 2 days. To quantify the effect of DHMBA on lipogenesis in 3T3-L1 cells, on day 0 and 2 days after differentiation culture, the differentiation medium contained either vehicle (final concentration 1% ethanol) or DHMBA (final concentrations 0.1, 1, 10, or 100 μM). After cell culture, the cells attached to the 24-well plate were gently washed twice with PBS and fixed with 4% formaldehyde dissolved in PBS for 30 minutes. Then, the fixed cells were washed three times with PBS, and Oil Red O solution (1.5% Oil Red O in isopropanol) was added and stained for 60 minutes. Panel A of Figure 4 is an explanatory diagram of the image of the stained lipid-accumulating cells observed under a microscope and photographed after staining. Panel B of Figure 4 is an explanatory diagram for quantifying the lipid (triglyceride) content. After Oil Red staining, the cells were extracted with isopropanol, and the absorbance of each well was measured at 510 nm using a spectrophotometer (μQuant, Bio-Tek Instruments). The data were obtained from 8 wells of two replicate culture plates using different cell preparations and are expressed as mean ± standard deviation. In addition, compared with the control group without DHMBA (gray bars), p < 0.001, indicating a significant difference. The significant difference test was based on one-way ANOVA and Tukey-Kramer post hoc test. [Figure 5] is an explanatory diagram showing the effect of the marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) on lipogenesis during the differentiation of 3T3-L1 mouse preadipocytes. The 3T3-L1 preadipocytes (1 x 10 per well of a 96-well plate) 4(Cells / ml) were cultured in DMEM medium containing 10% BCS and 1% P / S for 3 days until nearly confluent. Panel A of Figure 5 shows that after nearly confluence (day 0 of differentiation culture), the medium was replaced with DMEM medium containing 10% fetal bovine serum (FBS), 1% P / S, 0.5 mM IBMX, 1 μM dexamethasone, and insulin, with the addition of vehicle (final concentration 1% ethanol) or DHMBA (final concentrations 0.1, 1, 10, or 100 μM). After 2 days of culture, the medium was changed to fresh medium containing 10% FBS and insulin (10 μg / ml) (DMEM), with the addition of vehicle (final concentration 1% ethanol) or DHMBA (final concentrations 0.1, 1, 10, or 100 μM) for further 2 days of culture. Panel B of Figure 5 shows that after nearly confluence (day 0 of differentiation culture), the medium was replaced with a medium containing 10% fetal bovine serum (FBS), 1% P / S, 0.5 mM IBMX, 1 μM dexamethasone, insulin (10 μg / ml), and DHMBA (0.1, 1, 10, or 100 μM), and the cells were further cultured for 2 days. After 2 days of differentiation induction culture, the medium was changed to fresh medium containing 10% FBS and insulin (10 μg / ml) (without DHMBA), and the cells were further cultured for 2 days. Panel C of Figure 5 shows that after nearly confluence (day 0 of differentiation culture), the cells were cultured for 2 days in a differentiation-inducing factor-containing medium with 10% fetal bovine serum (FBS), 1% P / S, 0.5 mM IBMX, 1 μM dexamethasone, and insulin (10 μg / ml). Additionally, after 2 days of differentiation induction culture, the medium was changed to fresh medium containing 10% FBS, insulin (10 μg / ml), and DHMBA (0.1, 1, 10, or 100 μM), and the cells were further cultured for 2 days. After cell culture, the intracellular lipid formation was measured using a commercially available AdipoRed assay kit (Lonza, Walkersville, MD, USA). Fluorescence was measured with a 96-well plate reader using an excitation wavelength of 485 nm and an emission wavelength of 570 nm. Data were obtained from 8 wells in two replicate culture plates using different cell preparations and are presented as mean ± standard deviation. Additionally, compared to the control group without DHMBA (gray bars), p < 0.001, indicating a significant difference. The significance test was based on one-way ANOVA and Tukey-Kramer post hoc test. [Figure 6] shows a diagram explaining the effect of the marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) on adipogenesis of 3T3-L1 mouse preadipocytes in the presence of various inhibitors of the signaling pathway. 3T3-L1 preadipocytes (1x10 per well in a 96-well plate) 4The cells (1×10 cells / ml) were cultured in DMEM medium containing 10% BCS and 1% P / S for 3 days until they were nearly confluent. At the start of cell differentiation culture after near confluence (day 0 of differentiation induction culture), the medium was changed to DMEM containing 10% FBS, 1% P / S, 0.5 mM IBMX, 1 μM dexamethasone, and insulin (10 μg / ml), and the cells were further cultured for 2 days. Two days after this differentiation induction culture, the medium was replaced with fresh medium containing genistein (1 or 10 μM) (A in Fig. 6), Wortmannin (10 or 100 nM) (B in Fig. 6), PD98059 (1 or 10 μM) (C in Fig. 6), or Bay 11-7082 (1 or 10 nM) (D in Fig. 6), and vehicle (1% ethanol at the final concentration) or DHMBA (1 or 10 μM at the final concentration) was added, and the cells were further cultured for 2 days. After cell culture, the amount of intracellular lipid triglyceride formation was measured using the AdipoRed assay reagent (Lonza, Walkersville, MD, USA). Fluorescence was measured with a 96-well plate reader using excitation light at 485 nm and emission light at 570 nm. Data were obtained from 8 wells of two replicate culture plates using different cell preparations and are expressed as the mean ± standard deviation. In addition, compared with the control group without DHMBA (gray bars), p < 0.001, indicating a significant difference. The significance test was based on one-way ANOVA and Tukey-Kramer post hoc test. [Fig. 7] is an explanatory diagram showing the effect of the marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) on the concentrations of various proteins related to the signal transduction of fatty acid biosynthesis in adipocytes. The 3T3-L1 preadipocytes (1×10 6Cells (100 mm culture dish, 10 ml culture medium) were cultured in DMEM medium containing 10% BCS and 1% P / S for 3 days until nearly confluent. At the start of cell differentiation induction (day 0 of differentiation induction), the medium was replaced with DMEM medium containing 10% FBS, 1% P / S, 0.5 mM IBMX, and 1 μM dexamethasone, and the cells were cultured for an additional 2 days. After 2 days of culture, the medium was replaced with DMEM medium containing 10% FBS and insulin (10 μg / ml), DHMBA (final concentration 10 μM), and the cells were cultured for a further 2 days. After culture, cell lysates were prepared. Protein samples from the centrifuged supernatant were used at 40 μg of protein per lane for Western blot analysis. Panel A of Figure 7 shows representative data. In Panel B of Figure 7, the bars represent the fold change relative to the control group (without DHMBA). The data were obtained from different cell preparations and measured from a total of 4 membranes, and are presented as the mean ± standard deviation. In addition, compared with the control group, p < 0.01, indicating a significant difference. The significance test was based on one-way ANOVA and Tukey-Kramer post hoc test. [Figure 8] is a diagram explaining the mechanism by which 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) inhibits adipogenesis in 3T3-L1 adipocytes. DHMBA reduces the concentrations of the transcription factors PPARγ and C / EBPα, which are involved in the differentiation process of preadipocytes. In addition, DHMBA also reduces the concentrations of PI3K, Akt, MAPK, phosphorylated MAPK, and mTOR, which are involved in the process of promoting adipogenesis related to insulin signaling transduction. It is also explained that DHMBA inhibits adipogenesis regulated by the NF-κB signaling pathway. Thus, DHMBA exhibits an anti-adipogenic effect by regulating various signaling pathways in adipocytes.

Claims

1. Use of a 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) for the manufacture of a regulator for modulating signaling pathways in adipocytes, wherein the signaling pathways are C / EBPα, PI3K, Akt, MAPK, phosphorylated MAPK, mTOR, or NF-κB signaling pathways, and the regulator strongly modulates the signaling pathways in adipocytes in later stages of adipocyte differentiation compared to the early stages of adipocyte differentiation.

2. Use of a 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) for the manufacture of a reducing agent for reducing the concentration of proteins associated with the signaling process of adipogenesis in adipocytes, wherein the protein is C / EBPα, PI3 kinase 100α, Akt, MAPK, phosphorylated MAPK, mTOR, or NF-κB p65, wherein the reducing agent strongly reduces the concentration of proteins associated with the signaling process of adipogenesis in the later stages of adipocyte differentiation compared to the early stages of adipocyte differentiation.

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