Anti-obesity agent containing intestinal bacteria as active ingredient

Abstract

This anti-obesity agent is characterized by containing L. plantarum or the membrane vesicle (MV) thereof as an active ingredient.

Description

Anti-obesity agent with intestinal bacteria as an active ingredient

[0001] The present invention relates to an anti-obesity agent containing intestinal bacteria as an active ingredient.

[0002] Obesity is a condition in which the body becomes abnormally obese due to excessive fat deposition, and is one of the most prevalent diseases worldwide. Obesity also has the potential to promote diabetes, hyperlipidemia, hypertension, etc., making it a disease that requires immediate treatment.

[0003] Recent advances in DNA analysis and the establishment of enterobacteriaceae have revealed that changes in the intestinal environment are closely related to the maintenance of homeostasis in the host. For example, Akkermansia muciniphila (hereinafter referred to as "Am") is an enterobacterium that uses mucin as its sole nutrient source and has been reported to be associated with obesity (Non-Patent Document 1).

[0004] Gut, Vol. 65, pp. 426-436, 2016

[0005] However, no knowledge has yet been obtained about the relationship between intestinal bacteria other than Am and obesity, and there is still room for further research and development.

[0006] Therefore, an object of the present invention is to elucidate the mechanism of action of intestinal bacteria having anti-obesity activity and to provide a novel anti-obesity agent based on this mechanism of action.

[0007] As a result of intensive research, the present inventors have unexpectedly discovered that Lactiplantibacillus plantarum (hereinafter referred to as "L. plantarum" or "Lp") or its membrane vesicles (hereinafter referred to as "MV") serve as the active ingredient of an anti-obesity agent, and have elucidated its mechanism of action. Specifically, the characteristics of the anti-obesity agent of the present invention are as follows:

[0008] [1] An anti-obesity agent containing membrane vesicles (MVs) of L. plantarum as an active ingredient. [2] The anti-obesity agent according to [1] above, which is an agent for inhibiting adipocyte differentiation, inhibiting fat accumulation, or reducing accumulated fat. [3] An agent for promoting beige formation in adipocytes, containing L. plantarum or its membrane vesicles as an active ingredient. [4] The beige formation promoter according to [3] above, which has at least one effect selected from the inhibition of fat accumulation in adipocytes, increasing basal body temperature, and preventing and / or treating diabetes. [5] The beige formation promoter according to [3] above, which contains L. plantarum or its membrane vesicles as an active ingredient and is labeled with a label indicating that it has at least one effect selected from "making the body less prone to gain weight," "increasing basal body temperature," and "preventing and / or treating diabetes," or a label indicating that it is used to achieve said effect.

[0009] According to the present invention, an anti-obesity agent containing L. plantarum or its membrane vesicles (MVs) as an active ingredient can be provided.

[0010] FIG. 1 is a schematic diagram of adipocyte differentiation. FIG. 2 is a schematic diagram of the action of membrane vesicles (MVs). FIG. 3 is an explanatory diagram of the time schedule for testing anti-obesity effects. FIG. 4(A) is a diagram showing the differentiation-inhibitory effect of sLpMV on 3T3-L1 adipocytes. FIG. 4(B) is a diagram showing the differentiation-inhibitory effect of sLpMV on 3T3-L1 adipocytes. FIG. 4(C) is a diagram showing the differentiation-inhibitory effect of sLpMV on 3T3-L1 adipocytes. FIG. 5(A) is a diagram showing the fat accumulation-inhibitory effect of sLpMV on 3T3-L1 adipocytes. FIG. 5(B) is a diagram showing the fat accumulation-inhibitory effect of sLpMV on 3T3-L1 adipocytes. FIG. 6(A) is a diagram showing the fat-reducing effect of sLpMV on 3T3-L1 hypertrophied adipocytes. Figure 6(B) is a diagram showing the fat-reducing effect of sLpMV on 3T3-L1 hypertrophied adipocytes. Figure 7(A) is a diagram showing the differentiation-inhibitory effect of dLpMV on 3T3-L1 adipocytes. Figure 7(B) is a diagram showing the fat accumulation-inhibitory effect of dLpMV on 3T3-L1 adipocytes. Figure 7(C) is a diagram showing the fat-reducing effect of dLpMV on 3T3-L1 hypertrophied adipocytes. Figure 8 is a bar graph showing the time course of each gene in 3T3-L1 cells treated with sLpMV immediately after differentiation. Figure 9 is a bar graph showing the time course of each gene in 3T3-L1 cells treated with sLpMV after fat hypertrophy. Figure 10 is a diagram showing the mechanism of action of LpMV on fat accumulation inhibition. Figure 11 is a diagram showing the mechanism of action of LpMV on fat reduction.

[0011] The anti-obesity agent of the present invention containing intestinal bacteria as an active ingredient will be described in detail below. The following description of the constituent elements is an example of one embodiment of the present invention, and the present invention is not limited to these contents.

[0012] The present inventors have unexpectedly discovered that L. plantarum or its membrane vesicles (MVs) can be used as an active ingredient in anti-obesity agents, and have further elucidated its mechanism of action. When used as an anti-obesity agent, L. plantarum may be in the form of live bacteria, killed bacteria such as heat-killed bacteria (hereinafter referred to as "HKB"), or membrane vesicles (MVs). From the standpoint of size, membrane vesicles of L. plantarum are more preferred.

[0013] Furthermore, the present inventors have demonstrated that L. plantarum or its membrane vesicles, unlike other intestinal bacteria, have the effect of inhibiting the differentiation of precursor cells into adipocytes, inhibiting fat accumulation in adipocytes, and reducing accumulated fat in hypertrophied adipocytes. Furthermore, they have demonstrated that L. plantarum or its membrane vesicles have the effect of promoting the beige color of adipocytes.

[0014] (1. Anti-obesity Agent) In the present application, the term "anti-obesity agent" refers to an agent that, after being absorbed into the body, has an action of inhibiting differentiation into adipocytes, an action of inhibiting fat accumulation, or an action of reducing fat. The anti-obesity agent of the present application contains L. plantarum or its membrane vesicles (MVs) as an active ingredient.

[0015] The anti-obesity agent may contain components other than the L. plantarum bacteria to the extent that the effects of the present invention are not impaired. Examples of components other than the L. plantarum bacteria include medium components, solvents such as water, carbohydrates, proteins, lipids, vitamins, minerals, biologically essential trace metals (manganese sulfate, zinc sulfate, magnesium chloride, potassium carbonate, etc.), bacteria other than the L. plantarum bacteria, probiotics, and pharmaceutically acceptable carriers.

[0016] When the anti-obesity agent is an oral preparation, it can be in the form of a solid preparation such as a tablet, powder, fine granules, granules, capsules, pills or sustained-release preparation, or a liquid preparation such as a solution, suspension or emulsion.

[0017] In the present invention, the anti-obesity agent may be a pharmaceutical composition. When the pharmaceutical composition is formulated, acceptable additives may be used in combination. Examples of additives include excipients, stabilizers, preservatives, wetting agents, emulsifiers, lubricants, sweeteners, colorants, flavorings, buffers, antioxidants, and pH adjusters.

[0018] The amount of the anti-obesity agent to be taken is not particularly limited as long as the effects of the present invention are achieved, and can be appropriately adjusted depending on the age, health condition, weight, etc. of the person taking the anti-obesity agent.

[0019] For example, the active ingredient, L. plantarum, is used as the lower limit, and the dose is 1 x 10 ２ or more, more preferably 1×10 ６ It is recommended to take at least 1 x 10 per day. １５ or less, more preferably 1 x 10 １１ It is recommended to consume less than one serving.

[0020] If the daily intake is below the lower limit, the effect as an anti-obesity agent may be reduced, whereas if the daily intake is above the upper limit, the effect as an anti-obesity agent may reach a plateau and the effect per intake may not be achieved.

[0021] The anti-obesity agent is ideally taken continuously for a long period of time until obesity is eliminated. In order to maximize its effect, the lower limit is, for example, preferably 4 weeks or more, and more preferably several months to several years or more. If the dose is below the lower limit, the effect as an anti-obesity agent may be weakened.

[0022] When taking an anti-obesity agent continuously for a long period of time, there may be cases where the user neglects to take the agent due to unavoidable circumstances such as travel or work. In such cases, the frequency of intake may be adjusted as appropriate, for example, taking the agent 1 to 3 times a week, or taking the agent 2 to 3 times a day after leaving a gap of several days.

[0023] The subjects for taking the anti-obesity agent of the present invention are not particularly limited, but examples include those suffering from obesity, diabetes, hyperlipidemia, hypertension, lifestyle-related diseases, etc., or those in need of prevention of such diseases.

[0024] The present anti-obesity agent not only has the effect of treating the above diseases, but also has the effect of alleviating or ameliorating the worsening of symptoms and the effect of preventing the above diseases.

[0025] (2. L. plantarum) L. plantarum, the active ingredient of anti-obesity agents, is a gram-positive bacillus belonging to the genus Lactiplantibacillus, a type of human intestinal bacterium, and is used in many fermented foods. Among the active ingredients of Lactiplantibacillus plantarum, Lactiplantibacillus plantarum YIT 0132 (FERM BP-11349) is particularly preferred. Lactiplantibacillus plantarum YIT 0132 (old classification: Lactobacillus plantarum YIT 0132) was internationally deposited as Lactobacillus plantarum YIT 0132 (deposit number FERM BP-11349) on February 24, 2010, at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NITE-NPMD) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818). In recent years, lactic acid bacteria of the genus Lactobacillus have been reclassified. In other words, lactic acid bacteria that previously belonged to the genus Lactobacillus have been subdivided, and the genus names of some of the bacterial species have been changed.

[0026] (Regarding the reclassification of lactic acid bacteria) - Zheng et al., A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. Int. J. Syst. Evol. Microbiol. 2020 Apr; 70(4):2782-2858DOI 10.1099 / ijsem.0.004107 Therefore, in this specification, the new classification after the reclassification will be used. Furthermore, for example, lactic acid bacteria that were classified as Lactobacillus plantarum in the old classification but can now be classified as Lactiplantibacillus plantarum are included in Lactiplantibacillus plantarum in the present application.

[0027] When used as an anti-obesity agent, L. plantarum may be in the form of live cells, heat-killed cells (HKB) or other killed cells, or membrane vesicles (MVs). From the viewpoint of size, membrane vesicles are preferred. Regarding membrane vesicles, they will be explained separately later.

[0028] Heat-killed bacteria (HKB) are live bacteria killed by subjecting them to heat treatment for a predetermined period of time. Live and killed bacteria may be subjected to processes such as concentration / dilution, freezing, drying, powdering, and crushing. The heat treatment is not particularly limited as long as the effects of the present invention are achieved, and is carried out under conditions typically used to sterilize live bacteria. Furthermore, heat treatment is only one example of a method for killing bacteria, and the method for killing bacteria is not limited to heat treatment.

[0029] (3. Adipocytes) Adipocyte differentiation will be explained using Figure 1. Adipocytes consist of small adipocytes 103 and hypertrophic adipocytes 104, which are small adipocytes 103 that have accumulated excess lipid droplets due to excessive calorie intake or lack of exercise and have increased in size. Both types of adipocytes are produced by the differentiation of precursor cells 102 that are generated from mesodermal stem cells 101.

[0030] When the number and size of adipocytes increase, the volume of adipose tissue increases, resulting in the onset of obesity symptoms. In addition, the enlarged adipocytes 104 produce harmful adipocytokines, which cause insulin resistance in the body, resulting in the promotion of diabetes, hyperlipidemia, hypertension, etc.

[0031] Therefore, when a substance inhibits the process of differentiation of precursor cells 102 into small adipocytes 103, the substance acts as an inhibitor of adipocyte differentiation and can be considered as an active ingredient of an anti-obesity agent.

[0032] Furthermore, if the substance inhibits the process in which small fat cells 103 accumulate excess lipid droplets to form enlarged fat cells 104, it can be considered to be an active ingredient of an anti-obesity agent that acts as a fat accumulation inhibitor.

[0033] Furthermore, if a specific substance has the effect of reducing lipid droplets that have already accumulated in enlarged fat cells 104, that substance can be considered an active ingredient of an anti-obesity agent that acts as an agent for reducing accumulated fat.

[0034] (3.1 Beigeification) Mammals, including humans, have two main types of fat cells: white fat cells, which store and release fat as an energy source, and brown fat cells, which break down fat to generate energy.

[0035] In recent years, it has been reported that the expression of certain genes induces beige (brown) transformation of white fat cells, which, like brown fat cells, breaks down fat and suppresses fat accumulation. However, there are still many unknowns regarding the factors that induce the expression of certain genes.

[0036] As a result of extensive research, the present inventors have elucidated the mechanism by which L. plantarum or its membrane vesicles (MVs) acts as one of the factors that promote beige coloration.

[0037] In addition, by promoting the beige transformation of white adipocytes, it has the effects of suppressing fat accumulation in adipocytes, increasing basal body temperature, and preventing and / or treating obesity and diabetes.

[0038] Therefore, when implementing the present invention, functions and effects such as "making the body less prone to gaining weight," "raising basal body temperature," and "preventing obesity / diabetes" can be displayed on the product.

[0039] (4. Membrane Vesicles) Membrane vesicles (MVs) will be explained with reference to Figure 2. MVs 202 are vesicles produced by living enterobacteria 201. The average particle size of MVs is generally around 100 nm.

[0040] MV202 may be artificially produced as long as it exhibits the effects of the MVs disclosed in this invention. In this application, MVs isolated from the culture supernatant of L. plantarum are defined as sLpMVs, and MVs artificially produced from L. plantarum using a reagent such as deoxycholic acid (DOC) are defined as dLpMVs.

[0041] When produced from intestinal bacteria 201, MVs 202 are formed from the membrane of intestinal bacteria 201. Intestinal bacteria 201 generally have a size of about 1 μm, and therefore the intestinal bacteria themselves do not pass through the intercellular spaces of the intestinal epithelium 203. Instead, MVs 202 or membrane components of MVs 202 pass through the intercellular spaces and act on adipocyte precursor cells 102, small adipocytes 103, hypertrophied adipocytes 104, etc. (located at a distance) via blood vessels 204, etc. Intestinal bacteria 201 also function in intestinal homeostasis by acting on T cells 206, etc. via dendritic cells 205.

[0042] When used as an anti-obesity agent or a beige formation promoter, L. plantarum may be in the form of live cells, killed cells such as heat-killed cells (HKB), or membrane vesicles (MV). The anti-obesity agent can be produced by first culturing L. plantarum in step a, and then obtaining cells from the culture medium cultured in step a (step b1), or by centrifuging the culture medium cultured in step a and filtering the supernatant to obtain MV (step b2).

[0043] Instead of step b2, the culture medium may be centrifuged, the supernatant removed, and then treated with a deoxycholic acid (DOC) solution. After centrifuging again, the supernatant is filtered to obtain MVs (step b3), thereby producing an anti-obesity agent. The MVs produced through step b2 are sLpMVs, and the MVs produced through step b3 are dLpMVs.

[0044] EXAMPLES The present invention will be specifically described based on the following examples, but the present invention is not limited to these examples.

[0045] Example 1 <Intestinal Bacteria> L. plantarum YIT 0132 (FERM BP-11349) (hereinafter referred to as "Lp0132") was used as L. plantarum. Frozen Lp0132 was used and was subcultured and statically cultured in MRS medium at 37°C (hereinafter referred to as Lp0132 bacterial cell culture).

[0046] (Example 2) <Cells> The mouse fibroblast cell line 3T3-L1 (hereinafter referred to as "3T3-L1") used was cryopreserved. 3T3-L1 is characterized by the accumulation of lipid droplets within the cells upon induction of differentiation into adipocytes, and is therefore widely used as a cellular model system for lipid metabolism.

[0047] 3T3-L1 cells were cultured in a DMEM medium containing 10% FBS and 100 μg / mL streptomycin (10% FBS / DMEM) under 5% CO ２ Subculture was carried out at 37°C under the following conditions.

[0048] Example 3 Isolation of MVs (sLpMVs) The Lp0132 cell culture was centrifuged (8,000 xg, 20 min, 4°C) to remove the cells, and the culture supernatant was collected.

[0049] The collected culture supernatant was passed through a 0.45 μm PES syringe filter (Millipore) and then concentrated to approximately 1 mL using ultrafiltration (Millipore) with a molecular weight cutoff of 100 kDa. A density gradient was prepared using OptiPrep (60% iodixanol, Sigma-Aldrich).

[0050] The OptiPrep solution was diluted with MQ water, and 2 mL of 25% to 40% iodixanol solution was layered at 5% intervals. 1 mL of the concentrated supernatant was layered on top. Ultracentrifugation (P40ST rotor, 200,000 × g, 3 hours, 4°C) was performed, and 1 mL of the supernatant containing the band of interest was collected with a pipette. A solution of Lp0132 MVs (sLpMVs) isolated from the culture supernatant was obtained.

[0051] Example 4 Preparation of Heat-Killed Bacteria Cells Cells removed from the Lp0132 cell culture solution by centrifugation were suspended in MQ water and then centrifuged (10,000 × g, 10 min, 4°C) to wash the cells. This procedure was repeated three times, and then the tube containing the cells suspended in MQ water was placed in boiling water at 100°C for 10 minutes for heating. The heated cells were freeze-dried and powdered to prepare heat-killed cells (LpHKB).

[0052] Example 5 Preparation of MVs (dLpMVs) using deoxycholic acid (DOC) The pH of the Lp0132 cell culture was adjusted to 7.0 8 hours after the start of culture, and the culture was allowed to continue for 48 hours. The culture was then centrifuged (8,000 × g, 20 min, 4°C) and the culture supernatant was removed. The collected cells were washed with PBS and suspended in First Buffer (0.1 M Tris-HCl, 0.01 M EDTA (pH 8.9) solution).

[0053] After the bacterial precipitate had completely disappeared, Second Buffer (10% DOC solution) was added and stirred at room temperature for 30 minutes using a stirrer. The DOC-treated solution was centrifuged (12,000 × g, 30 minutes, 4 ° C.) and the supernatant was collected. The collected supernatant was passed through a 0.45 μm PES syringe filter (Millipore) and then concentrated to approximately 1 mL by ultrafiltration (Millipore) with a molecular weight cutoff of 100 kDa.

[0054] OptiPrep (60% iodixanol, Sigma-Aldrich) was used to prepare the density gradient. The OptiPrep solution was diluted with MQ water, and 1 mL of 10% to 40% iodixanol solution was layered at 5% intervals. 1 mL of the concentrated supernatant was layered on top. This was ultracentrifuged (P40ST rotor, 200,000 × g, 3 h, 4°C), and 1 mL of the desired band was collected with a pipette. A solution of Lp0132 MV (dLpMV) prepared by DOC was obtained.

[0055] MV protein was quantified by a predetermined method using Pierce™ BCA Protein Assay Kit (Thermo), and the amount of MV was converted into the amount of protein.

[0056] Example 6 Anti-obesity effect of sLpMV Figure 3 shows the time schedule for Example 6. The horizontal axis represents the time elapsed since the start of culture, and each schematic diagram on the horizontal axis represents the process of changes in 3T3-L1 cells during culture. Predetermined amounts of a differentiation inducer, insulin, and MV were added to 3T3-L1 cells, and after the start of culture, fatty oil droplets were stained with Oil Red O and observed on days 3, 10 (Point A), 24 (Point B), and 34 (Point C). Details are as follows.

[0057] (Example 6.1) <Differentiation-inhibitory effect> To examine whether sLpMV has an inhibitory effect on adipocyte differentiation, 3T3-L1 cells were cultured for 2 days after addition of 5 μg / mL or 10 μg / mL of sLpMV along with the differentiation inducers dexamethasone, 3-isobutyl-1-methylxanthine, and insulin.

[0058] Furthermore, the medium was replaced with insulin-containing 10% FBS / DMEM containing sLpMV and cultured for two days, after which the medium was replaced with regular insulin-containing 10% FBS / DMEM medium (without MV or HKB), and on the third day the cells were subjected to analysis of intracellular oil droplet amounts and adipocyte-related gene expression levels.

[0059] Example 6.1.1 Measurement of Intracellular Oil Droplet Amount Using Oil Red O The culture supernatant of 3T3-L1 cells cultured in a 24-well plate was removed, and the cells were washed once with PBS. After that, 10% neutral buffered formalin was added and the cells were left to stand overnight at 4°C to fix the cells. Thereafter, the cells were washed three times with MQ water, and Oil Red O staining solution (Cosmo Bio) from the Lippitt assay kit was added, followed by staining at room temperature for 1 hour.

[0060] After staining, the cells were washed several times with MQ water until the color of the Oil Red O staining solution disappeared. After washing, the cells were dried at 37°C, and the stained cell images were photographed under a microscope. The stained 3T3-L1 cells were added with the extract solution (Cosmo Bio) from the Lippitt assay kit and left for 30 minutes to extract Oil Red O. After extraction, the absorbance (505 nm) of the extract solution was measured using a plate reader to quantify the amount of intracellular oil droplets in the 3T3-L1 cells. In other examples, when measuring the amount of intracellular oil droplets using Oil Red O, the same method as in Example 6.1.1 was used.

[0061] Example 6.1.2 Gene Expression Analysis by Real-Time PCR 3T3-L1 cells cultured in a 24-well plate using the above-described culture method were harvested, and total RNA was purified using an Illustra RNAspin kit (Cytiva) according to the prescribed method. The purified RNA was reverse transcribed using GoScript™ Reverse Transcriptase (Promega), and real-time PCR was performed using GoTaq® qPCR Master Mix (Promega) and a QuantStudio™ 12K Flex Real-Time PCR System (Applied Biosystems) according to the accompanying operating instructions.

[0062] The primers used were designed from known mRNA sequences using Primer Express Software Version 3.0 (Applied Biosystems) (see Table 1). Gene expression analysis by real-time PCR in other examples was also performed using the same method as in Example 6.1.2, and all of the primers used are listed in Table 1.

[0063]

[0064] The characteristics of the genes related to the primers shown in Table 1 are briefly explained as follows: PPAR-γ (peroxisome proliferator-activated receptor gamma) is a master regulator of adipocyte differentiation. Adiponectin is a protein secreted from differentiated adipocytes. ATGL (Adipose triglyceride lipase) activates the breakdown of fatty oil droplets. HSL (Hormone-sensitive lipase) activates the breakdown of fatty oil droplets. UCP-1 (Uncoupling protein 1) promotes heat production in mitochondria. PGC-1α (peroxisome proliferators-activated receptor-γ coactivator-1α) enhances mitochondrial biogenesis and increases UCP-1 expression. CGI-58 (comparative gene identification-58) functions as a cofactor for ATGL.

[0065] Figures 4(A) to 4(C) show the differentiation-inhibitory effect of sLpMV on 3T3-L1 progenitor cells. Figure 4(A) shows Oil Red O staining images (corresponding to point A in Figure 3) of 3T3-L1 cells cultured for 7 days after adding various concentrations of sLpMV and inducing differentiation for 2 days. From left to right, the cells are untreated, treated with 5 μg / mL of sLpMV, and treated with 10 μg / mL of sLpMV. Compared to the control, cells treated with sLpMV showed weaker staining with Oil Red O, confirming that the cells have a concentration-dependent inhibitory effect on differentiation.

[0066] Figure 4(B) is a bar graph in which the amount of lipid droplets was quantified based on absorbance of Oil Red O extracted from the stained cells shown in Figure 4(A). The horizontal axis shows, from left to right, the untreated control, 5 μg / mL sLpMV, and 10 μg / mL sLpMV. The vertical axis shows the amount of intracellular lipid droplets (% Control).

[0067] The p values ​​are as follows: *: p<0.05, **: p<0.01, ***: p<0.001. The same explanation applies to the subsequent graphs.

[0068] From FIG. 4(B), it was confirmed that the amount of lipid oil droplets extracted and quantified was significantly less in cells to which sLpMV had been added.

[0069] Figure 4 (C, left) shows the expression level of PPAR-γ mRNA. The horizontal axis shows, from left to right, the untreated control, sLpMV 5 μg / mL, and sLpMV 10 μg / mL. The vertical axis shows the expression level of PPAR-γ mRNA. The PPAR-γ mRNA level in each cell is corrected by the mRNA expression level of β-actin as an internal standard.

[0070] PPAR-γ is a protein belonging to the nuclear receptor superfamily and is a master regulator that is expressed during differentiation into adipocytes. The degree of differentiation of 3T3-L1 cells can be confirmed by measuring the expression level of PPAR-γ mRNA.

[0071] From FIG. 4 (C, left), it was confirmed that the expression level was reduced by the addition of sLpMV, and that sLpMV suppresses differentiation into adipocytes.

[0072] Figure 4 (C right) shows the expression level of adiponectin mRNA. The horizontal axis shows, from left to right, the untreated control, sLpMV 5 μg / mL, and sLpMV 10 μg / mL. The vertical axis shows the expression level of adiponectin mRNA. The adiponectin mRNA level in each cell is shown as the result of correction using the mRNA expression level of β-actin as an internal standard.

[0073] Adiponectin is a type of adipocytokine, a physiologically active protein secreted by differentiated adipocytes. The degree of differentiation of 3T3-L1 cells can be confirmed by measuring the expression level of adiponectin mRNA.

[0074] FIG. 4 (right C) also shows that the expression level was reduced by the addition of sLpMV, confirming that sLpMV suppresses differentiation into adipocytes.

[0075] (Example 6.2) <Fat accumulation inhibitory effect> Next, adipocytes were treated with sLpMV for 21 days starting immediately after differentiation to examine the effect of sLpMV on the process of transformation into hypertrophic adipocytes. Figures 5(A) and 5(B) show the effect of sLpMV on fat accumulation inhibitory effect on 3T3-L1 adipocytes.

[0076] 5(A) shows images of Oil red O staining of cells cultured with sLpMV for 21 days after differentiation induction and untreated cells (corresponding to point B in FIG. 3). From left to right, the cells are an untreated control, 5 μg / mL of sLpMV, and 10 μg / mL of sLpMV added. Compared to the control, cells treated with sLpMV showed weaker Oil red O staining, confirming that sLpMV has a concentration-dependent effect of inhibiting fat accumulation.

[0077] Figure 5(B) is a bar graph in which the amount of lipid droplets was quantified by absorbance of Oil Red O extracted from the stained cells shown in Figure 5(A). The horizontal axis shows, from left to right, the untreated control, 5 μg / mL sLpMV, and 10 μg / mL sLpMV. The vertical axis shows the amount of intracellular lipid droplets (% Control).

[0078] Figure 5(B) confirms that the amount of intracellular lipid droplets in adipocytes continuously treated with sLpMV was significantly 30 to 40 percent lower than in untreated controls, indicating that fat accumulation was suppressed.

[0079] (Example 6.3) <Fat-reducing effect on hypertrophic adipocytes> Next, after differentiation into adipocytes, the cells were cultured for 21 days, and the effect of sLpMV on the hypertrophic adipocytes was examined. After hypertrophy, the cells were cultured for an additional 10 days in media with or without sLpMV, and the amount of lipid oil droplets contained in the hypertrophic adipocytes was measured. Figures 6(A) and 6(B) show the fat-reducing effect of sLpMV on hypertrophic adipocytes in 3T3-L1 adipocytes.

[0080] Figure 6(A) shows an image of Oil red O staining (corresponding to point C in Figure 3) of enlarged cells cultured for 21 days after differentiation induction and treated with sLpMV for 10 days. From left to right, there is an untreated control, 5 μg / mL of sLpMV, and 10 μg / mL of sLpMV were added. Compared to the control, the red staining with Oil red O in enlarged adipocytes treated with sLpMV is lighter, confirming the fat-reducing effect.

[0081] Figure 6(B) is a bar graph in which the amount of lipid droplets was quantified by absorbance of Oil Red O extracted from the stained cells shown in Figure 6(A). The horizontal axis shows, from left to right, the untreated control, 5 μg / mL sLpMV, and 10 μg / mL sLpMV. The vertical axis shows the amount of intracellular lipid droplets (% Control).

[0082] As shown in FIG. 6(B), a reduction of about 10 to 20% in fat mass was observed in the hypertrophied adipocytes treated with sLpMV, confirming the effect of sLpMV in reducing fat accumulation in hypertrophied adipocytes.

[0083] Example 7 Anti-obesity effect of dLpMV The anti-obesity effect study shown in Example 6 was carried out using MVs (sLpMV) prepared from the culture supernatant of Lp0132 in Example 3. Therefore, when artificially prepared dLpMV was used in Example 4, a comparison was made with sLpMV to determine whether it exhibited similar anti-obesity properties against 3T3-L1.

[0084] In Example 7, the anti-obesity effect was investigated in the same manner as in Example 6, except that sLpMV was replaced with dLpMV (prepared in Example 5) or LpHKB (prepared in Example 4).

[0085] Figures 7(A) to 7(C) show the differentiation-inhibitory effect of dLpMV on 3T3-L1 adipocytes (Figure 7(A)), fat accumulation-inhibitory effect (Figure 7(B)), and fat-reducing effect on hypertrophic adipocytes (Figure 7(C)). Figure 7(A) corresponds to the test results in Figure 4(B), Figure 7(B) corresponds to the test results in Figure 5(B), and Figure 7(C) corresponds to the test results in Figure 6(B).

[0086] 7(A) to 7(C), the horizontal axis represents, from left to right, the untreated control, dLpMV 5 μg / mL, dLpMV 10 μg / mL, and LpHKB 10 μg / mL, and the vertical axis represents the amount of intracellular oil droplets (% Control).

[0087] Figures 7(A) to 7(C) show that, like sLpMV, dLpMV also inhibited the differentiation of 3T3-L1 cells into adipocytes (Figure 7(A)), inhibited fat accumulation (Figure 7(B)), and reduced fat in hypertrophied adipocytes (Figure 7(C)), and that the strength of these effects was comparable.

[0088] Furthermore, although LpHKB also had the effect of inhibiting differentiation into adipocytes and fat accumulation, LpMV was found to have a stronger effect. This is thought to be due to the difference in the contact surface area between HKB and adipocytes, as HKB is much larger than MV.

[0089] Example 8: <Investigation of the mechanism of anti-obesity effect of LpMV> Based on the results above, it was confirmed that LpMV has an anti-obesity effect. Therefore, in Example 8, the mechanism of action of LpMV in inhibiting fat accumulation and reducing fat in enlarged adipocytes was investigated using sLpMV.

[0090] First, to investigate the mechanism of accumulation suppression, the cells were collected on days 7, 14, and 21 of sLpMV treatment, and the expression levels of PPAR-γ, adiponectin mRNA, and the mRNA levels of lipid metabolism-related genes ATGL, HSL, CGI-58, UCP-1, and PGC-1α in the adipocytes were examined.

[0091] 8 is a bar graph showing the time course of each gene in 3T3-L1 cells treated with sLpMV immediately after differentiation. Each graph, from top left to bottom right, shows the mRNA expression changes of the adipocyte-related gene PPAR-γ, adiponectin, and the lipid metabolism-related genes ATGL, HSL, CGI-58, UCP-1, and PGC-1α. The horizontal axis shows the time course (day 7, day 14, day 21), and the vertical axis shows the mRNA expression level of each gene. The mRNA level of each gene is shown as a result of correction using the mRNA expression level of the internal standard β-actin. Furthermore, each bar graph for each group, from left to right, shows the untreated control, sLpMV 5 μg / mL, and sLpMV 10 μg / mL.

[0092] 8, while little change was observed in the expression levels of PPAR-γ and adiponectin mRNA, it was confirmed that the expression levels of ATGL and CGI-58 mRNA increased in cells treated with sLpMV on day 21. Furthermore, the expression levels of UCP-1 and PGC-1α mRNA increased compared to the control from day 7 of sLpMV treatment, suggesting the possibility that lipid metabolic pathways in adipocytes were activated.

[0093] Next, to investigate the mechanism of the fat-reducing effect of sLpMV on hypertrophic adipocytes, adipocytes were cultured for 21 days after differentiation to hypertrophy, and then the hypertrophic adipocytes treated with sLpMV for 5 and 10 days were collected and the mRNA expression levels of PPAR-γ, adiponectin gene, and lipid metabolism-related genes ATGL, HSL, CGI-58, UCP-1, and PGC-1α were examined.

[0094] 9 is a bar graph showing the time course of each gene in 3T3-L1 treated with sLpMV after lipohypertrophy. Each graph, from top left to bottom right, shows the mRNA expression changes of the adipocyte-related gene PPAR-γ, the adiponectin gene, and the lipid metabolism-related genes ATGL, HSL, CGI-58, UCP-1, and PGC-1α. The horizontal axis shows the time course (day 5, day 10), and the vertical axis shows the mRNA expression level of each gene. The mRNA level of each gene shows the results corrected for the mRNA expression level of the internal standard β-actin. Furthermore, each bar graph for each group, from left to right, shows the untreated control, sLpMV 5 μg / mL, and sLpMV 10 μg / mL.

[0095] 9 shows that there was a tendency for increased expression of the adiponectin gene and PPAR-γ mRNA on day 5 after treatment, and a significant increase in PPAR-γ mRNA on day 10. Furthermore, while a significant increase was observed in the mRNA of lipid metabolism-related genes ATGL, HSL, and CGI-58, no increase was observed in the mRNA of UCP-1 and PGC-1α in hypertrophied adipocytes, suggesting that the fat accumulation-inhibiting effect on adipocytes and the fat-reducing effect on hypertrophied adipocytes are exerted by different mechanisms.

[0096] <Mechanism of action of LpMV for the fat accumulation inhibitory effect> To summarize the results of fat accumulation inhibition in Example 8, treatment with sLpMV showed almost no change in the expression levels of PPAR-γ and adiponectin mRNA in cells (FIG. 8).

[0097] Furthermore, the mRNA expression of ATGL and CGI-58, genes involved in the breakdown of lipid droplets, increased on day 21 in cells treated with sLpMV, whereas the mRNA expression of UCP-1 and PGC-1α was significantly increased at the early stage on day 7 of sLpMV treatment (Figure 8).

[0098] Here, PGC-1α is a molecule identified as a transcriptional coactivator that binds to PPAR-γ, but PGC-1α alone also controls the expression of many genes involved in energy production and heat consumption.

[0099] It is known that when PGC-1α is introduced into white fat cells, mitochondrial biogenesis is enhanced and UCP-1 expression increases, regardless of the PPAR-γ gene, resulting in the cells becoming beige fat cells that actively burn fat.

[0100] In other words, while PPAR-γ mRNA did not change in adipocytes treated with sLpMV, expression of UCP-1 and PGC-1α mRNA was high, and fat accumulation was suppressed even when cultured in high glucose. This suggests that sLpMV has the effect of promoting beige adipocyte-like transformation of adipocytes through induction of PGC-1α and UCP-1 gene expression.

[0101] The inventors have confirmed that MVs from Alistipes indistinctus (hereinafter referred to as "AiMV"), an intestinal bacterium different from L. plantarum, have the same fat accumulation inhibitory effect as L. plantarum. However, the fat accumulation inhibitory effect of AiMV was suggested to be due to increased expression of the PPAR-γ gene, which in turn increased expression of UCP-1 and ATGL and HSL genes, which are responsible for decomposing fatty oil droplets.

[0102] In other words, it was revealed that LpMV has a different mechanism of action for inhibiting fat accumulation than AiMV. Figure 10 is a schematic diagram showing the mechanism of action of LpMV for the aforementioned inhibitory effect on fat accumulation.

[0103] <Mechanism of action of LpMV on fat-reducing effect> Next, to summarize the fat-reducing effect results of Example 8, when sLpMV was treated after induction of hypertrophic adipocytes, no change was observed in UCP-1 and PGC-1α mRNA, while PPAR-γ mRNA increased, and accordingly, ATGL, HSL, and CGI-58 mRNA increased.

[0104] In other words, it was revealed that, like AiMV, it reduces fat in enlarged adipocytes by promoting fat breakdown.

[0105] From the above, it has become clear that the effect of LpMV on adipocytes is different from that on hypertrophic adipocytes. Figure 11 is a schematic diagram showing the mechanism of action of LpMV on fat reduction.

[0106] These results demonstrate that L. plantarum membrane vesicles (MVs) are an active ingredient in anti-obesity agents. Furthermore, L. plantarum membrane vesicles (MVs) are effective as adipocyte differentiation inhibitors, fat accumulation inhibitors, or fat accumulation reduction agents, and a new mechanism of action (e.g., the mechanism of action for promoting beige formation) has been discovered. Furthermore, it has been confirmed that L. plantarum functions effectively as an anti-obesity agent not only in the form of membrane vesicles but also when added as bacterial cells such as HKB.

[0107] 101 Stem cells 102 Progenitor cells 103 Small adipocytes 104 Hypertrophic adipocytes 201 Intestinal bacteria 202 Membrane vesicles (MVs)

Claims

1. An anti-obesity agent containing membrane vesicles (MVs) of L. plantarum as an active ingredient.

2. The anti-obesity agent according to claim 1, which is an agent for inhibiting adipocyte differentiation, an agent for inhibiting fat accumulation, or an agent for reducing accumulated fat.

3. An agent for promoting beige formation in adipocytes, containing L. plantarum or its membrane vesicles as an active ingredient.

4. The beige formation promoter according to claim 3, which has at least one effect selected from the group consisting of inhibiting fat accumulation in adipocytes, increasing basal body temperature, and preventing and / or treating diabetes.

5. The beige formation promoter according to claim 3, which contains L. plantarum or membrane vesicles thereof as an active ingredient and is labeled to have at least one effect selected from "making the body less prone to gaining weight," "increasing basal body temperature," and "preventing and / or treating diabetes," or is labeled for use in achieving said effect.

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