Composition for preventing or treating diabetes comprising novel bacillus wiedmannii MBT09 strain and culture thereof as active ingredient

Abstract

The present invention relates to a composition for preventing or treating diabetes, comprising a novel Bacillus Wiedmannii MBT09 strain and a culture thereof as an active ingredient. According to the present invention, unlike rosiglitazone, which only targets PPARγ to increase various types of insulin-responsive proteins, stimulation was carried out through a pathway that encompasses both the activation of PPARγ through ERK and the increase of the PI3K / AKT pathway, which is a downstream regulatory pathway targeting IRS that initiates by binding to insulin receptors, to synthesize various types of insulin-responsive proteins, and thus it was concluded that insulin-dependent cellular signaling pathways are significantly activated.

Description

A composition for preventing or treating diabetes comprising a novel Bacillus widmannii MBT09 strain and a culture thereof as an active ingredient

[0001] The present invention relates to a composition for preventing or treating diabetes, comprising a novel Bacillus widmannii MBT09 strain and a culture thereof as an active ingredient.

[0002] Diabetes is a disease that remains incompletely conquered. Due to various environmental and genetic factors, it remains difficult to treat or cure, resulting in significant casualties worldwide. Type 2 diabetes, a metabolic disease, accounts for 90% of all diabetes-related mortality and leads to complications such as diabetes-related heart disease and stroke, making it a disease worthy of attention.

[0003] In other words, diabetes is a chronic condition characterized by persistent hyperglycemia that disrupts not only carbohydrate metabolism but also lipid and protein metabolism. Its pathological manifestations are diverse, and those directly attributable to hyperglycemia include diabetic peripheral neuropathy, diabetic retinopathy, diabetic nephropathy, diabetic cataracts, keratopathy, and diabetic arteriosclerosis, all of which affect the retina, kidneys, nerves, and cardiovascular systems.

[0004] A major problem with current diabetes treatments is the side effects of existing medications. Treatment options include sulfonylureas, biguanides, alpha-glucosidase inhibitors, thiazolidinediones, and glinides. Rosiglitazone, the thiazolidinedione control used in this study, works by increasing insulin sensitivity in peripheral tissues such as adipose tissue and muscle, thereby improving insulin resistance. Side effects include increased triglyceride levels and an increased risk of cardiovascular disease, myocardial infarction, macular edema, and fractures. The development of new treatments that can address these issues is urgently needed.

[0005] The present inventors identified Bacillus WiedmanniiMBT09, a microorganism that showed the highest PPARγ agonist effect on 3T3-L1 preadipocytes among 105 types of microorganisms, and isolated the active ingredient, Cyclo(L-Pro-L-Try). They demonstrated that the binding of receptors activated by Bacillus WiedmanniiMBT09 and Cyclo(L-Pro-L-Try) increased lipid-related markers and insulin-dependent pathways such as IRS, PI3K, and AKT, and simultaneously increased GLUT4 present in the cell membrane.

[0006] The present invention provides a novel Bacillus Wiedmannii MBT09 strain, deposited under accession number KCCM13378P.

[0007] The above strain culture is characterized by increasing the differentiation rate of adipocytes.

[0008] The above strain culture is characterized by increasing the expression of PPARγ, AP2, LPL, CD36, FAS, C / EBPα, FAS, IRS-1, and PI3K genes.

[0009] The above strain culture is characterized by upregulating the insulin signaling pathway.

[0010] The above strain culture is characterized by increasing the expression of IRS, PI3K, p-AKT / AKT, and p-ERK / ERK genes.

[0011] The above strain culture is characterized by increasing AKT or GLUT4 gene expression.

[0012] The above strain culture is characterized by increasing intracellular glucose uptake rate and reducing cellular insulin resistance.

[0013] According to another embodiment of the present invention, a pharmaceutical composition for preventing or treating diabetes is provided, which comprises the strain or a culture solution of the strain as an active ingredient.

[0014] According to another embodiment of the present invention, a food composition for improving diabetes is provided, which comprises the strain or a culture solution of the strain as an effective ingredient.

[0015] According to another embodiment of the present invention, a pharmaceutical composition for preventing or treating diabetes is provided, comprising a compound of [chemical formula 1] as an active ingredient;

[0016] [Chemical Formula 1]

[0017] .

[0018]

[0019] Unlike Rosiglitazone, which targets only PPARγ and increases various insulin-responsive proteins, the present invention stimulates the synthesis of various insulin-responsive proteins through a pathway encompassing both the activation of PPARγ through ERK and the increase of the PI3K / AKT pathway, a downstream regulatory pathway, by targeting IRS, which is initiated by binding to the insulin receptor, thereby significantly activating the insulin-dependent cell signaling pathway. Therefore, the microorganisms Bacillus Wiedmannii MBT09 and Cyclo (L-Pro-L-Try) can be used as anti-diabetic therapeutic agents.

[0020] Figure 1 is a graph showing the cytotoxic effect on 3T3-L1 cells after treatment with MCS (50, 100, or 200 μg / mL) for eight selected microorganisms as a result of screening for microorganisms with antidiabetic effects on 3T3-L1 cells.

[0021] Figure 2 is a summary diagram of the MCS of the B. wiedmanniiMBT09 active ingredient purification method.

[0022] Figure 3 is a graph showing cell viability in the presence of (A) B. wiedmanniiMBT09 and (B) cyclo(L-pro-L-tyr).

[0023] Figure 4 shows the effects of B. wiedmannii MBT09 and cyclo(L-pro-L-tyr) on adipocyte differentiation; (A) Adipocyte differentiation rate using B. wiedmannii MBT09. (B) Adipocyte differentiation rate using cyclo(L-pro-L-tyr). (C) Fluorescence microscopy image. 3T3-L1 cells were treated with B. wiedmannii MBT09 for a 5-day differentiation induction period.

[0024] Figure 5 shows the effect of MCS of B. wiedmanniiMBT09 on diabetes biomarkers in 3T3-L1 cells. 3T3-L1 cells were treated with MCS of B. wiedmanniiMBT09 (20, 100, or 500 μg / mL).

[0025] Figure 6 shows the effect of MCS of B. wiedmanniiMBT09 on the activation of the insulin signaling pathway in 3T3-L1 cells. (A, C) Effect of MCS of B. wiedmanniiMBT09 on the protein expression of IRS, PI3K, p-AKT, AKT, p-ERK, ERK, and PPARγ in 3T3-L1 cells. (B) Schematic diagram of biomarkers related to the insulin signaling pathway.

[0026] Figure 7 shows the effect of MCS of B. wiedmanniiMBT09 on the activation of the glucose signaling pathway in 3T3-L1 cells. (A, C) Effect of MCS of B. wiedmanniiMBT09 on the protein expression of p-AKT, AKT, and GLUT4 in 3T3-L1 cells. (B) Schematic diagram of biomarkers related to the insulin signaling pathway.

[0027] Figure 8 is a graph showing the effect of cyclo(L-pro-L-tyr) on diabetes biomarkers in 3T3-L1 cells. 3T3-L1 cells were treated with cyclo(L-pro-L-tyr) at 2, 20, or 200 μg / mL. mRNA expression levels were determined using qRT-PCR.

[0028] Figure 9 shows the effect of cyclo(L-pro-L-tyr) on the activation of the insulin signaling pathway in 3T3-L1 cells. (A, B) Effect of cyclo(L-pro-L-tyr) on the protein expression of IRS, PI3K, p-AKT, AKT, p-ERK, ERK, and PPARγ in 3T3-L1 cells.

[0029] Figure 10 shows the effect of Cyclo(L-Pro-L-Try) on the activation of the glucose signaling pathway in 3T3-L1 cells. (A, B) Effect of Cyclo(L-Pro-L-Try) on the protein expression of p-AKT, AKT, and GLUT4 in 3T3-L1 cells.

[0030] In the case of Fig. 11, it is a graph showing the 2-NBDG absorption effect of B. wiedmannii MBT09 culture solution and Cyclo(L-pro-L-tyr).

[0031] The present invention provides a novel Bacillus Wiedmannii MBT09 strain, deposited under accession number KCCM13378P.

[0032] According to another embodiment of the present invention, a pharmaceutical composition for preventing or treating diabetes is provided, which comprises the strain or a culture solution of the strain as an active ingredient.

[0033] According to another embodiment of the present invention, a food composition for improving diabetes is provided, which comprises the strain or a culture solution of the strain as an effective ingredient.

[0034] According to another embodiment of the present invention, a pharmaceutical composition for preventing or treating diabetes is provided, comprising a compound of [chemical formula 1] as an active ingredient;

[0035] [Chemical Formula 1]

[0036] .

[0037] The present invention is described in more detail through the following examples. However, the present invention is not limited to these examples and may be embodied in any form. The examples presented are provided to aid understanding of the present invention.

[0038]

[0039] The present invention provides a novel Bacillus Wiedmannii MBT09 strain deposited under the accession number KCCM13378P. The strain stimulates the synthesis of various proteins that respond to insulin through a pathway encompassing both the activation of PPARγ through ERK and the increase of the PI3K / AKT pathway, a downstream regulatory pathway targeting IRS that is initiated by binding to the insulin receptor, thereby significantly activating the insulin-dependent cell signaling pathway.

[0040] The above strain culture is characterized by increasing intracellular glucose uptake rate and reducing cellular insulin resistance, and therefore, the strain or strain culture can be used for the prevention or treatment of diabetes.

[0041] The term "culture" of the present invention means the entire medium including the strain, bacterial extract, metabolites thereof, and extra nutrients obtained by culturing the Bacillus Widmannii MBT09 strain (Accession No.: KCCM13378P) for a certain period of time in a medium capable of supplying nutrients so that the strain can grow and survive, but also includes a culture solution from which the strain has been removed after culturing the strain.

[0042] The above culture solution may be left to stand for a certain period of time, and only the upper liquid may be taken, excluding the portion that has settled to the lower layer, or the bacterial cells may be removed through filtration, or the culture solution may be centrifuged to remove the lower sediment and only the upper liquid may be taken. This culture solution may also be concentrated using a conventional method and used as a concentrate.

[0043] The above culture solution or concentrate of the culture solution can be dried by a conventional method and provided as a dried product.

[0044] The term "culture" as used herein, unless otherwise specified, includes a culture solution of the aforementioned fungi, a concentrate of the culture solution, or a dried product of the culture solution or concentrate. The culture may or may not contain fungi, and the inclusion or absence of fungi is not particularly problematic.

[0045] The above strain culture is characterized by increasing the differentiation rate of adipocytes and increasing the expression of PPARγ, AP2, LPL, CD36, FAS, C / EBPα, FAS, IRS-1, and PI3K genes. PPARγ overexpression promotes adipocyte development in 3T3-L1 cells, whereas PPARγ inhibition inhibits adipogenesis. In other words, it was found that the strain regulates factors related to insulin-dependent gene expression of adipocytes and plays an important role in intracellular fatty acid and triglyceride metabolism.

[0046] The above strain culture is characterized by increasing IRS, PI3K, p-AKT / AKT, and p-ERK / ERK gene expression, which means that it upregulates the insulin signaling pathway.

[0047] Additionally, the strain culture is characterized by increased AKT or GLUT4 gene expression. AKT not only plays a crucial role in glucose regulation, but also induces altered responses between insulin sensitivity and downstream oxidative metabolism, leading to insulin secretion. This finding suggests that the strain upregulates the glucose signaling pathway in cells.

[0048] According to another embodiment of the present invention, a pharmaceutical composition for preventing or treating diabetes is provided, which comprises the strain or a culture solution of the strain as an active ingredient.

[0049] According to another embodiment of the present invention, a food composition for improving diabetes is provided, which comprises the strain or a culture solution of the strain as an effective ingredient.

[0050] According to another embodiment of the present invention, a pharmaceutical composition for preventing or treating diabetes is provided, comprising a compound of [chemical formula 1] as an active ingredient;

[0051] [Chemical Formula 1]

[0052] .

[0053] The compound of the above [chemical formula 1] stimulates the synthesis of various types of proteins that respond to insulin through a pathway encompassing both the activation of PPARγ through ERK and the increase of the PI3K / AKT pathway, which is a downstream regulatory pathway targeting IRS that is initiated by binding to the insulin receptor, thereby significantly activating the insulin-dependent cell signaling pathway.

[0054] The compound of the above [chemical formula 1] is characterized by increasing the intracellular glucose uptake rate and reducing the insulin resistance of the cell, and therefore, the compound of the above [chemical formula 1] can be used for the prevention or treatment of diabetes.

[0055] The compound of the above [chemical formula 1] is characterized by increasing the differentiation rate of adipocytes and increasing the expression of PPARγ, AP2, LPL, CD36, FAS, C / EBPα, FAS, IRS-1, and PI3K genes. PPARγ overexpression promotes adipocyte development in 3T3-L1 cells, whereas PPARγ inhibition inhibits adipogenesis. In other words, it was found that the compound of the above [chemical formula 1] regulates factors related to insulin-dependent gene expression of adipocytes and plays an important role in intracellular fatty acid and neutral fat metabolism.

[0056] The compound of the above [chemical formula 1] is characterized by increasing the expression of IRS, PI3K, p-AKT / AKT, and p-ERK / ERK genes, which means that it upregulates the insulin signaling pathway.

[0057] Furthermore, the compound of [Chemical Formula 1] is characterized by increasing AKT or GLUT4 gene expression. AKT not only plays a crucial role in glucose regulation, but also induces altered responses between insulin sensitivity and downstream oxidative metabolism, leading to insulin secretion. This finding suggests that the compound of [Chemical Formula 1] upregulates the glucose signaling pathway in cells.

[0058] The pharmaceutical composition for preventing or treating diabetes can be prepared as a composition in a conventional dosage form by selecting one or more active ingredients selected from the group consisting of the Bacillus Wiedmannii MBT09 (BACILLUS WIEDMANNIIMBT09) strain of the present invention (Accession No.: KCCM13378P) as the main ingredient, a culture of the strain, a concentrate of the culture, a dried product of the culture, an extract of the culture, a dead cell of the strain, and a compound of [Chemical Formula 1] in an effective amount, and one or more pharmaceutically acceptable conventional carriers or one or more additives.

[0059] The carrier may be selected from one or more of diluents, lubricants, binders, disintegrants, sweeteners, stabilizers, and preservatives, and the additive may be selected from one or more of flavorings, vitamins, and antioxidants.

[0060] In the present invention, any pharmaceutically acceptable carrier and additive may be used. Specifically, as a diluent, lactose monohydrate, trehalose, cornstarch, soybean oil, microcrystalline cellulose, or D-mannitol is preferable, as a lubricant, magnesium stearate or talc is preferable, and as a binder, polyvinyl pyrrolidone (PVP) or hydroxypropylcellulose (HPC) is preferable. In addition, it is preferable to select from among carboxymethylcellulose calcium (Ca-CMC), sodium starchglycolate, polacrylin potassium, or cross-linked polyvinylpyrrolidone as a disintegrant, white sugar, fructose, sorbitol, or aspartame as a sweetener, carboxymethylcellulosesodium (Na-CMC), β-cyclodextrin, white bee's wax, or xanthan gum as a stabilizer, and methyl p-hydroxy benzoate (methylparaben), propyl p-hydroxybenzoate (propylparaben), or It is preferable to choose from potassium sorbate, but it is not limited thereto.

[0061] The pharmaceutical composition of the present invention can be administered to a patient as a single dose, or can be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. The term "pharmaceutically effective amount" as used herein refers to an amount that produces a greater response than a negative control group, and preferably refers to an amount sufficient to prevent or treat an inflammatory disease. In addition, the pharmaceutically effective amount may be appropriately varied depending on various factors, such as the disease and its severity, the patient's age, weight, health status, sex, route of administration, and treatment period.

[0062] The composition of the present invention can be formulated in various ways according to the route of administration using a method known in the art together with the pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable" refers to a non-toxic composition that is physiologically acceptable, does not inhibit the action of the active ingredient when administered to humans, and does not typically cause allergic reactions such as gastrointestinal disorders or dizziness or similar reactions. The composition of the present invention can be formulated in various ways according to the route of administration using a method known in the art together with the pharmaceutically acceptable carrier. The route of administration is not limited thereto, but may be administered orally or parenterally.

[0063] According to another embodiment of the present invention, a food composition for preventing diabetes is provided, which comprises as an active ingredient at least one selected from the group consisting of Bacillus Wiedmannii MBT09 strain (Accession No.: KCCM13378P), a culture of the strain, a concentrate of the culture, a dried product of the culture, an extract of the culture, dead cells of the strain, and a compound of [Chemical Formula 1].

[0064] According to another embodiment of the present invention, a health functional food composition for preventing diabetes is provided, which comprises as an active ingredient at least one selected from the group consisting of Bacillus Wiedmannii MBT09 strain (Accession No.: KCCM13378P), a culture of the strain, a concentrate of the culture, a dried product of the culture, an extract of the culture, dead cells of the strain, and a compound of [Chemical Formula 1].

[0065] The health functional food of the present invention may be a beverage (including tea or alcoholic beverages), fruit and processed foods thereof (e.g., canned fruit, bottled fruit, jam, marmalade, etc.), fish, meat and processed foods thereof (e.g., ham, sausage, corned beef, etc.), bread and noodles (e.g., udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (e.g., butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein, retort food, frozen food, various seasonings (e.g., soybean paste, soy sauce, sauce, etc.), etc., including the food composition.

[0066] In addition, the health functional food of the present invention may be formulated as a tablet, pill, powder, granule, powder, capsule, liquid, etc. These may be formulated by further including one or more of a carrier, diluent, excipient, and additive.

[0067] Specific examples of the carrier, excipient, diluent and additive include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil, and it is preferable to use at least one selected from the group consisting of:

[0068]

[0069] <Experimental Method>

[0070] 1. Cell viability analysis

[0071] To determine the cytotoxicity of Bacillus wiedmannii culture, MTT reagent was used. 3T3-L1 cells were cultured at 1X10 4 Cells were plated in 96-well plates per well. After overnight incubation, the medium was replaced, and samples were dispensed at various concentrations. After 48 hours of incubation, 10 μL of a 5 mg / mL MTT stock solution was added to each well, and the samples were incubated at 37°C for 2 hours. After removing the medium, 100 μL of DMSO was added to each well. Formazan was dissolved in DMSO and detected at 540 nm using a microplate reader (TECAN, Mannedorf, Switzerland).

[0072]

[0073] 2. 2-NBDG absorption analysis

[0074] Glucose uptake into adipocytes was measured using a modified experiment. Briefly, C2C12 cells were allowed to reach 100% confluence for 3 days. Cell differentiation was confirmed 2 days after differentiation medium treatment. Cells were cultured in serum-free medium for 24 h and then treated with 2-NBDG (50 μM) for 24 h in the presence or absence of B. wiedmannii MBT09, cyclo(L-pro-L-tyr). Insulin (100 nM) was used as a positive control for glucose uptake. 2-NBDG uptake was stopped by washing the cells twice with phosphate-buffered saline. The remaining fluorescence activity of the cell monolayer was recorded at 485 and 535 nm using a fluorescence microplate reader. The fluorescence activity in the absence of 2-NBDG was subtracted from all values ​​obtained.

[0075]

[0076] 3. Open column chromatography

[0077] Bacillus wiedmanniiMBT09 was cultured in tryptic soy broth autoclaved at 121°C for 15 min in a 3-L 9-L fermentor. The culture conditions were as follows: 37°C for 4 h; 100 rpm; pH, 6.9–7.2. After 48 h, the MCS of B. wiedmannii was centrifuged at 3,000 rpm for 20 min and filtered. The filtered MCS was extracted three times with ethyl acetate at room temperature. After removing the undissolved residue, the ethyl acetate extract was concentrated in vacuo to obtain a dry residue. The residue was then applied to chromatography on a SiO2 column (Φ 4 × 20 cm), eluting with chloroform / methanol (10:1) → chloroform / methanol / water (30:3:1). The second column used an ODS column (Φ 2 × 7 cm) containing acetone / water (1:1) → (2:1). The third column used SiO2 column (Φ 2 × 12 cm) chromatography eluted with chloroform / methanol / water (30:3:1). The biologically active fraction was determined according to the AdipoRed assay in 3TL-1 cells, and the TLC pattern was monitored using a UV Spectronic Co. (Westbury, NY, USA).

[0078]

[0079] 4. Animal testing

[0080] (1) Experimental diet and animal husbandry

[0081] Seven-week-old male dbdb mice (Laon Biotech Animal Co., Ltd., Seoul, Korea) were purchased as experimental animals and acclimatized for one week before being divided into five groups (one normal group, three test groups, and one positive control group). Three animals were assigned to each group, and the experiment was conducted for five weeks. The breeding conditions of the animal laboratory were maintained at 22.0±1.0℃, humidity 50.0%±10.0%, and a 12-h light / dark cycle. The mice were fed a basic feed without any quantity restriction, and drinking water was measured at 200 g and 250 mL, respectively. Body weight was recorded once a week at a set time, and food and water intake were also measured at a set time every day.

[0082]

[0083] (2) Measurement of body weight, water consumption, and food consumption

[0084] Body weights of experimental animals were measured once a week for five weeks, starting from the start of the experiment. Food and water intake were measured once a day during the experimental period. Food and water intake were measured based on residual amounts from the previous intake.

[0085]

[0086] (3) Changes in fasting blood sugar levels

[0087] Blood glucose levels were measured four times at one-week intervals. After fasting for 6 hours, blood was collected from the tail vein of the experimental animals, and fasting blood glucose levels were measured using a blood glucose meter (Accu-Check Active; Roche Diagnostics GmbH, Mannheim, Germany).

[0088]

[0089] <Experimental Results>

[0090] 1. Screening of microorganisms with PPARγ agonistic effects on 3T3-L1 cells.

[0091] To select microorganisms with PPARγ agonistic effects on 3T3-L1 cells, cells were treated with 10 μg / mL of Microbial Culture Supernatant (MCS) for 5 days and analyzed using the AdipoRed assay. As a result, among 37 microorganisms, 8 MCS (has12, SMSR7, MHSMT5, M2YM8, M2YM10, SFT18, M2YT5, and SFT2) showed PPARγ agonistic effects. Microorganisms that specifically showed the effect of PPARγ agonists on 3T3-L1 cells were selected by treating the MCS of the 8 microorganisms at various concentrations (50, 100, and 200 μg / mL) (Fig. 1). Among them, SFT18 was the most effective.

[0092] To confirm the phylogeny of the above microorganism, the inventors analyzed the 16S rRNA gene sequence (Sequence Listing 1). Based on the 16S rRNA gene sequence analysis, the SFT18 strain was identified as Bacillus Wiedmannii and ultimately named Bacillus WiedmanniiMBT09. It was deposited with the Korea Microbiological Conservation Center on August 17, 2023, under the accession number KCCM13378P.

[0093] [Sequence List 1]

[0094]

[0095]

[0096] 2. Open column chromatography analysis

[0097] A 90% ethyl acetate extract (B. wiedmannii MBT09) was obtained from B. wiedmannii strain 9L. Overall, the inventors fractionated the ethyl acetate extract and isolated each compound according to the flow chart shown in Fig. 2. Among the ten first fraction samples, no. 7 was the most effective PPARγ agonist. In the second round, sample no. 1 (7-1) was the best, and in the third round, sample no. 4 (7-1-4) was the best. In addition, the active ingredient was purified based on the NMR results and was confirmed to be cyclo(L-pro-L-tyrosine) of [chemical formula 1];

[0098] [Chemical Formula 1]

[0099] .

[0100]

[0101] 3. Effect of B. wiedmanniiMBT09 and cyclo(L-pro-L-tyr) on 3T3-L1 cell viability

[0102] The MTT assay was used to determine the cell viability of B. wiedmannii and cyclo(L-pro-L-tyr) in 3T3-L1 cells cultured for 24 h, after incubation with the following samples at concentrations up to 500 (μg / mL) for 48 h. Within the tested concentration range (15.6–500 μg / mL), B. wiedmanniiMBT09-treated cells exhibited viability similar to that of untreated control cells (Fig. 3A). Furthermore, cyclo(Lpro-L-tyr) at the tested concentrations (6.25–200 μg / mL) showed viability similar to that of untreated control cells (Fig. 3B).

[0103]

[0104] 4. Effects of B. wiedmanniiMBT09 and cyclo(L-pro-L-tyr) on adipocyte differentiation

[0105] Based on the results of the MTT assay, three concentrations were set to lower the survival rate to 1 / 5 of the total concentration of 500. B. wiedmanniiMBT09 and cyclo(L-pro-L-tyr) were administered at concentrations of 20, 100, 500 and 25, 50, 100, 200, respectively. The effects of B. wiedmannii and cyclo(L-pro-L-try) on the differentiation rate are shown in Fig. 4A and B. Cells were stained with AdipoRed on day 5 to observe lipid droplets indicated by green areas in the cytoplasm.

[0106] On day 5, we found that the number and size of lipid droplets in the sample-treated groups (20, 100, and 500 μg / mL) increased compared to the control group, despite maintaining the normal phenotype and morphology of mature adipocytes (Fig. 4C). Furthermore, the sample-treated groups (20, 100, and 500 μg / mL and 25, 50, 100, and 200 μg / mL) showed a dose-dependent increase in adipocyte differentiation rate compared to the untreated group (Figs. A and B).

[0107]

[0108] 5. Effect of B. wiedmanniiMBT09 on gene expression of transcription factors in adipocytes.

[0109] Based on previous experimental results, messenger RNA (mRNA) expression levels were examined to elucidate the effects of PPARγ agonists, adipogenesis, lipogenesis, and insulin signaling pathways in 3T3-L1 cells induced by MCS of B. wiedmanniiMBT09.

[0110] After differentiation into adipocytes, the expression of PPARγ, a major target of PPARγ agonists, and FAS, LPL, CD36, AP2, and C / EBPα, which are downstream factors of adipogenesis, increased (Fig. 5). In addition, PI3K and IRS, which play important roles in the insulin signaling pathway, tended to increase dependently after B. wiedmannii MBT09 treatment (Fig. 5). PPARγ overexpression promotes adipocyte development in 3T3-L1 cells, whereas PPARγ inhibition inhibits adipogenesis. Based on this PPARγ pathway theory, total RNA was purified from differentiated 3T3-L1 cells and qRT-PCR was performed. These results suggest that the sample regulates factors related to insulin-dependent gene expression in adipocytes and plays an important role in intracellular fatty acid and triglyceride metabolism.

[0111]

[0112] 6. Upregulation of the insulin signaling pathway in 3T3-L1 cells by B. wiedmanniiMBT09 culture medium.

[0113] To investigate the effects of MCS of B. wiedmanniiMBT09 on the insulin signaling pathway in 3T3-L1 cells, we evaluated the expression levels of IRS, PI3K, p-AKT / AKT, p-ERK / ERK, and PPARγ by Western blot. Western blot analysis showed that MCS of B. wiedmanniiMBT09 (20, 100, or 500 μg / mL) increased the expression levels of the insulin receptor IRS-1; PI3K, a central regulator of insulin; and AKT, a regulator of energy metabolism. Adipose tissue also increased insulin-sensitizing PPARγ and its downstream factor p-ERK / ERK (Fig. 6). These findings suggest that MCS of B. wiedmanniiMBT09 upregulates the insulin signaling pathway in 3T3-L1 cells. Therefore, B. MCS of wiedmanniiMBT09 can exhibit antidiabetic effects in 3T3-L1 cells.

[0114]

[0115] 7. Upregulation of the glucose signaling pathway in 3T3-L1 cells by B. wiedmanniiMBT09 culture medium.

[0116] To investigate the effects of MCS from B. wiedmanniiMBT09 on the glucose signaling pathway in 3T3-L1 cells, we assessed the expression levels of p-AKT, AKT, and GLUT4 using Western blot analysis. Western blot analysis showed that treatment with MCS from B. wiedmanniiMBT09 (20, 100, and 500 μg / mL) increased the expression levels of p-AKT, AKT, and GLUT4 (Fig. 7). Furthermore, according to the above pathway, AKT not only plays a crucial role in glucose but also induces altered responses between insulin sensitivity and downstream oxidative metabolism of insulin secretion. These findings suggest that MCS from B. wiedmanniiMBT09 upregulates the glucose signaling pathway in 3T3-L1 cells. Therefore, MCS from B. wiedmanniiMBT09 may have antidiabetic effects in 3T3-L1 cells.

[0117]

[0118] 8. Effect of cyclo(L-pro-L-tyr) on gene expression of transcription factors in adipocytes

[0119] Based on the previous experimental results, we investigated messenger RNA (mRNA) expression levels to elucidate the effects of cyclo(L-pro-L-tyr)-induced MCS on PPARγ agonism, adipogenesis, lipogenesis, and the insulin signaling pathway in 3T3-L1 cells. After cyclo(L-pro-L-tyr) treatment, the expression of PPARγ, a central axis of PPARγ agonism, increased, as did the expression of IRS, a component of insulin signaling. Furthermore, the expression levels of LPL, AP2, and Cd36, which are downstream factors of adipogenesis, also increased, whereas the expression of FAS tended to decrease (Fig. 8). PPARγ overexpression promotes adipocyte development in 3T3-L1 cells, whereas PPARγ inhibition inhibits adipogenesis. Based on this PPARγ pathway theory, total RNA was purified from differentiated 3T3-L1 cells and qRT-PCR was performed.

[0120]

[0121] 9. Upregulation of the insulin signaling pathway in 3T3-L1 cells by Cyclo(L-pro-L-tyr)

[0122] IRS-1 is a PI3K receptor and plays a crucial role in insulin regulation. Western blot analysis was performed to determine whether the effects of cyclo(L-pro-L-tyr) on 3T3-L1 cells were related to this pathway. The activity of the insulin receptor IRS-1 was upregulated after cyclo(L-pro-L-tyr) treatment. This indicates that cyclo(Lpro-L-tyr) upregulates the phosphorylation of PI3K and AKT (downstream factors of IRS-1), leading to upregulation of the IRS-1, PI3K, and AKT pathways. Furthermore, in adipose tissue, PPARγ, which is associated with insulin sensitivity, and its downstream factor p-ERK / ERK were increased, and PPAR, which increases insulin-mediated glucose uptake and improves insulin resistance, increased in a dose-dependent manner (Figure 9). Therefore, cyclo(L-pro-L-tyr) can be considered an effective antidiabetic agent.

[0123]

[0124] 10. Upregulation of the glucose signaling pathway in 3T3-L1 cells by Cyclo(L-pro-L-tyr)

[0125] In adipocytes, GLUT4 translocation is accelerated by the pancreas via the PI3K / AKT signaling pathway. AMPK-mediated GLUT4 translocation is promoted by muscle contraction via a transient increase in intracellular calcium concentration. Based on this theory, factors related to the glucose uptake mechanism were identified as p-AKT / AKT, GLUT4, and p-AMPK / AMPK. Both AKT and PI3K, downstream factors of GLUT4, were increased, and AMPK, which phosphorylates independently of glucose metabolism and promotes fatty acid oxidation, also increased in a dose-dependent manner (Fig. 10). Therefore, cyclo(L-pro-L-tyr) is considered an effective antidiabetic agent.

[0126]

[0127] 11. Effect of B. wiedmanniiMBT09 culture medium and Cyclo(L-pro-L-tyr) on 2-NBDG uptake

[0128] Referring to Figure 11, it was confirmed that when 3T3-L1 cells and C2C12 cells were treated with B. wiedmanniiMBT09 culture medium and Cyclo(L-pro-L-tyr), the cellular sugar uptake was promoted (Figure 11). Therefore, it can be seen that B. wiedmanniiMBT09 culture medium and Cyclo(L-pro-L-tyr) exhibit a diabetes treatment effect by promoting sugar uptake into cells and thereby lowering blood sugar concentration.

[0129]

[0130] 12. Effects of B. wiedmanniiMBT09 on body weight, food intake, water consumption, and blood sugar levels

[0131] During the experimental period, body weight and drinking water were measured three times a week for four weeks. Statistical analysis revealed that, considering the NaCl-administered control group and the rosiglitazone-administered control group (Table 1), the experimental group's body weight was similar to that of the previous group and lower than that of the rosiglitazone-administered group. Second, there was no difference in diet between the control group and the sample group. Finally, regarding drinking water intake, the experimental group consumed a relatively smaller amount than the NaCl-administered control group, but a larger amount than the rosiglitazone-administered group.

[0132] To determine the effect of the sample on blood glucose levels, blood samples were collected from the tail vein of experimental animals once a week to measure blood glucose levels. After acclimation, the blood glucose levels of control db / db mice remained at 594 ± 5.6 from the start of the experiment until dissection.

[0133] On the other hand, the blood sugar concentrations of the sample treatment group (20, 100, 500) and the rosiglitazone administration group sequentially decreased to 541±18.7, 466±25.5, 480±16.3, and 386.4±56.05 (Table 1).

[0134] GroupFood consumption(g / week)Body weight Gain(g / week)Water consumption(g / week)Blood sugardbdb mouse (each n=3)Control 15.32 ± 4.530.26 ± 0.2167.6 ± 10.63594 ± 5.6ROSI(mg / kg / day)14.4 ± 3.691.15 ± 0.5814.7 ± 5.60386 ± 56.05MBT09(mg / kg / day)2016.4 ± 5.051.79 ± 0.3644.7 ± 15.7541 ± 18.7710016.4 ± 5.351.37 ± 0.3640 ± 15.4466.2 ± 25.5550014.5 ± 4.962.61 ± 0.635.7 ± 12.5480.6 ± 16.3

[0135]

[0136] Conclusion

[0137] In the present invention, we screened for effective microorganisms using 3T3-L1, a mouse progenitor cell line commonly used in diabetes research. Furthermore, when cells are differentiated into adipocytes by differentiation induction, the number of insulin receptors increases rapidly, which is used to measure changes in glucose uptake and insulin signaling in diabetes research. For screening, 37 out of 105 strains were selected in the first stage, and 9 strains were selected in the second stage (Fig. 1). Among them, SFT18, which showed the highest concentration-dependent efficacy during concentration treatment, was selected. The microbial base sequence and characteristics were confirmed using 16S rRNA, and it was identified as B. wiedmannii MBT09.

[0138] The present inventors identified pathways that can demonstrate the efficacy of diabetes-related insulin mechanisms and glucose uptake mechanisms through cytotoxicity, gene expression, and protein expression in 3T3-L1 cells using MCS of B. wiedmannii MBT09.

[0139] In addition, the active ingredient cyclo(L-pro-L-tyr) was isolated and confirmed from the microbial MCS (Fig. 2). The active ingredient obtained through isolation also confirmed the pathway of its mechanism of action through the same gene and protein expression as the MCS of B. wiedmanniiMBT09. First, the inventors examined IRS, which acts as an insulin receptor substrate; the insulin mechanism PI3K pathway activated by stimulation; and AKT, a downstream protein. Activation of AKT, which acts as an insulin-stimulated transduction pathway, dose-dependently increased ERK and PPARγ, which play a role in improving insulin resistance. Finally, Glut4, which acts as a glucose transporter in the step of regulating the glucose uptake rate, is an essential expression factor in the intracellular glucose uptake process and an essential component of the insulin signaling system, was also increased.

[0140] Based on these results, the difference between the existing diabetes treatment drug rosiglitazone and the microbial sample is that the former binds only to PPARγ to alleviate insulin resistance, whereas it directly and sequentially acts on IRS, PI3K, and AKT to increase insulin sensitivity. The difference is that the above pathway also has two gene transcription pathways, including the existing mechanism of rosiglitazone, which activates PPARγ through ERK as AKT increases. In type 2 diabetes, insulin resistance and a state requiring high amounts of insulin for normal glucose metabolism occur simultaneously, and glucose homeostasis is not maintained due to reduced insulin action. It is also a major cause of premature onset of diabetes. Diabetes is classified as the number one cause of death and accounts for 90% of diabetic patients. Insulin plays a crucial role in increasing tissue glucose utilization by inducing the binding of the insulin receptor to the IRS, translocation of GLUT4 through multiple pathways, and maintenance of stable blood glucose concentration. Based on these results, it was confirmed that adiponectin-related factors, insulin-related factors, and glucose-related factors involved in insulin resistance and diabetes are effective in improving insulin resistance and increasing glucose utilization.

[0141]

[0142] Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the following claims also fall within the scope of the present invention.

[0143]

[0144] [Accession number]

[0145] Name of depositor: Korea Center for Microbiological Conservation (KCCM)

[0146] Accession number: KCCM13378P

[0147] Date of acceptance: 20230817

[0148]

[0149] [Microorganism Deposit Certificate]

[0150]

Claims

1. A novel Bacillus Wiedmannii MBT09 strain deposited under accession number KCCM13378P.

2. In paragraph 1, The above strain culture solution is a Bacillus Widmannii MBT09 strain characterized by increasing the differentiation rate of adipocytes.

3. In paragraph 1, The above strain culture solution is characterized by increasing the expression of PPARγ, AP2, LPL, CD36, FAS, C / EBPα, FAS, IRS-1, and PI3K genes.

4. In paragraph 1, The above strain culture medium is a strain characterized in that it upregulates the insulin signaling pathway.

5. In paragraph 1, The above strain culture medium is characterized by increasing the expression of IRS, PI3K, p-AKT / AKT, and p-ERK / ERK genes.

6. In paragraph 1, The above strain culture medium is a strain characterized by increasing AKT or GLUT4 gene expression.

7. In paragraph 1, The above strain culture medium is characterized by increasing intracellular glucose uptake rate and reducing cellular insulin resistance.

8. A pharmaceutical composition for preventing or treating diabetes, comprising the strain of any one of claims 1 to 7, or a culture solution of the strain, as an active ingredient.

9. A food composition for improving diabetes comprising the strain of any one of claims 1 to 7, or a culture solution of the strain, as an active ingredient.

10. A pharmaceutical composition for preventing or treating diabetes, comprising a compound of [chemical formula 1] as an active ingredient; [Chemical Formula 1] .

Images