KR20140144496A - The products containing probiotic lactobacillus acidophilus HY7037 having activity preventing from insulin resistance, which caused type 2 diabetes mellitus - Google Patents

Abstract

The present invention relates to Lactobacillus acidophilus HY7037 having an effect of improving insulin resistance and a product containing the same as an active ingredient. Lactobacillus acidophilus HY7037 according to the present invention can improve an insulin resistance inducing environment by primarily increasing the glucose uptake reduced by TNF-α, increases gene and protein expressions of GLUT4 involved in the glucose uptake, increasing protein phosphorylation of AKT and IRS(Tyr)-1 of the insulin signal pathway influencing translocation and expression of GLUT4, and increasing the expression levels of PPARγ and PGC-1α known to be involved in insulin sensitivity, in L6 skeletal muscle cells of rats having insulin resistance induced by TNF-α, and can improve an insulin resistance environment which is mainly caused by type 2 diabetes by reducing the gene level of IL-6 increased by TNF-α, which is a kind of inflammatory cytokine, and thus can be variously used for a pharmaceutical composition for preventing and treating metabolic syndrome, a functional health food, a functional beverage, a fermented product, and the like.

Description

Lactobacillus acidophilus HY7037 having an insulin resistance improving effect and a product containing the same as an active ingredient (The products containing probiotic lactobacillus acidophilus HY7037 have an activity to prevent insulin resistance, which caused type 2 diabetes mellitus}

The present invention relates to a Lactobacillus acidophilus strain HY7037 having an insulin resistance improving effect and a product containing the same as an active ingredient. More particularly, the present invention relates to a Lactobacillus acidophilus strain HY7037 having an insulin resistance- , Increased GLUT4 gene expression and protein expression associated with glucose uptake, increased translocation of GLUT4, increased glucose uptake by TNF-a primarily in L6 skeletal muscle cells, And the expression levels of PPARγ and PGC-1α, which are known to be involved in insulin sensitivity, increase the protein phosphorylation of AKT and IRS-1 (Tyr) in the insulin signal pathway, To increase the induction of insulin resistance and to increase the expression level of IL-6 by TNF-α, a type of inflammatory cytokine. To type 2 Lactobacillus ash having an insulin resistant environment improved activity that is a main cause of diabetes is also pillar's (Lactobacillus acidophilus) HY7037 and Metabolic Syndrome for prevention and pharmaceutical composition containing them as an active component, fermented milk, beverage, and Functional food.

Insulin resistance is a symptom in which insulin moves to the target tissue through the blood and does not react to target tissues. It is a symptom of the loss of glucose and fat homeostasis due to insufficient blood glucose lowering and lipolysis, which are the main roles of insulin. Therefore, those who show insulin resistance have a high blood sugar level and a high level of insulin in their blood. If these symptoms continue, fat breakdown continues in the adipocytes, glucose uptake does not occur in the muscle, and glucose production continues in the liver. These symptoms continue to persist insulin resistance and obesity, fatty liver, diabetes, and so on. In recent years, many researches have been made to solve the cause of such insulin resistance and to improve the insulin resistance.

TNF (tumor necorsis factor) -a is a multipurpose cytokine, which is involved in immune regulation, apoptotic cell death, and tumor cell lysis. Recent studies have shown that TNF-α induces insulin resistance by inhibiting insulin signal transduction. TNF-α also promotes protein phosphorylation of IRS-1 (Ser). This results in the inhibition of IRS-1 (Tyr) protein phosphorylation by insulin, which in turn affects the signal pathway driven by insulin and ultimately uptakes glucose GLUT4 will also be affected. These TNF-α are expressed in adipose tissue and muscle tissue, and are also known to be expressed in animal models involved in obesity and insulin resistance.

In addition, PPARr and PGC-1α are low expression in muscle and adipose tissue of obesity and diabetic animal models through many existing papers, and they are also implicated in insulin sensitivity, and they are also proved to be deeply related to insulin resistance .

On the other hand, lactic acid bacterium, which exists in many fermented foods such as man's cabbage, kimchi and cheese, and milk and milk, plays a role of decomposing fibrous and complex proteins into important nutrients while symbiosis with the human digestive system. To inhibit the growth of harmful bacteria such as E. coli and Chlostridum sp., And to improve diarrhea and constipation, as well as to play a role in vitamin synthesis and blood cholesterol lowering. Especially lactic acid bacteria can bind strongly to the mucous membrane and epithelial cells of the intestines, which helps a lot of the action. Particularly, Lactobacillus acidophilus is one of the most widely distributed lactic acid bacteria in the natural world, and it has an acid resistance such as pH control of milk or a toxic substance produced when fermentation is performed and within the biliary and the steel, and has antibacterial activity against Puja Solarium (Fusarium), Pseudomonas (Pseudomonas), a useful strain that has effects such as antioxidant and anti-inflammatory, arteriosclerosis prevention.

Thus, the present inventors have found that Lactobacillus acidophilus HY7037 isolated from feces of a newborn about 7 days after birth is reduced by TNF-α in L6 skeletal muscle cells induced by insulin resistance by TNF-α The expression of GLUT4, which is involved in glucose uptake, and the expression of PPARγ and PGC-1α, which are involved in insulin sensitivity, are also increased, thereby promoting insulin signal transduction And has an activity of improving insulin resistance, thus completing the present invention.

Korean Patent Publication No. 2011-0101885 (Title of the invention: Composition for preventing or treating diabetic or diabetic complications comprising an effective ingredient of Dandelion Lactic Acid Fermentation Liquid, Disclosure Date: September 16, 2011) Korean Patent Laid-Open Publication No. 2012-0055828 (Name of the invention: Lactobacillus acidophilus strain K-59, K-59 strains for improving insulin secretion, and a method for producing the same) Work)

The present invention promotes IRS-1 (Tyr), AKT protein phosphorylation, which is reduced by TNF-α in L6 skeletal muscle cells of rat induced by insulin resistance by TNF-α, Lactobacillus acidophilus (GLUT4), which has the effect of increasing the expression of GLUT4 and the expression of PPARγ and PGC-1α genes, which are also involved in insulin sensitivity, to maintain insulin signaling and improve insulin resistance Lactobacillus acidophilus ) HY7037 and a pharmaceutical composition for preventing and treating metabolic syndrome containing the same as an active ingredient, fermented milk, beverage and health functional food.

In order to achieve the above object, the present invention provides a pharmaceutical composition for improving insulin signaling pathway of IRS-1 (Tyr) and AKT inhibited by TNF-α of L6 skeletal muscle cells induced by insulin resistance by TNF-α, Lactobacillus acidophilus HY7037, which has an effect of improving insulin resistance by increasing the expression of GLUT4 and insulin sensitivity related to PPARγ and PGC-1α, and preventing the metabolic syndrome containing the active ingredient And a therapeutic pharmaceutical composition, fermented milk, beverage and health functional food.

Hereinafter, the present invention will be described in detail.

In order to isolate the new strain according to the present invention, the feces collected from the neonate 7 days before birth were collected, and 0.1 g of the collected neonatal feces was decanted into the exhalation diluent to prepare a 1.5% agar-containing MRS ), Cultured at 37 ° C for 48 hours, colony was taken as a loop and inoculated on fresh MRS solid medium and purified. The purely isolated colonies were inoculated into the MRS liquid medium and cultured at 37 DEG C for 24 hours.

L6 skeletal muscle cells, which are the muscle cells of the rat induced by insulin resistance by TNF- [alpha], were tested for the effect of increasing blood glucose uptake in each of several single strains in the cultured newborn feces. Lactobacillus acidophilus HY7037 was selected.

In order to identify the newly isolated strains, the sugar fermentation experiment was performed using the API 50 CH kit of Biomerieux Co. The results are shown in Table 1 below.

As can be seen from the following Table 1, the sugar fermentation experiment of the new strain of the present invention showed that Lactobacillus acidophilus , which is 100% identical to the sugar fermentation experiment of Lactobacillus acidophilus ( Lactobacillus acidophilus ) acidophilus HY7037, deposited with KCTC 12409BP, Korea on May 9, 2013.

The characteristics of Lactobacillus acidophilus HY7037 according to the present invention are as follows.

1) Types of bacteria

The characteristics of the bacteria when cultured on an MRS agar plate medium at 37 ° C for 2 days

① Cell shape: Bacillus

② Mobility: None

③ Spore forming ability: None

④ Gram staining: positive

2) Morphology

When cultured on MRS agar plate medium at 37 ° C for 2 days,

① Shape: Circular

② Bump: convex

③ Surface: Smooth

3) Physiological properties

① Growth temperature: Growth possible Growth temperature 15 ~ 40 ℃

            Optimum growth temperature 37 ℃

② Growth pH: Growable growth pH 5.0 ~ 7.5

           Optimum pH 6.0 to 6.5

③ Effect on oxygen: Tumor anaerobic

4) Catalase: -

5) Whether gas is formed: -

6) Growth at 15 ℃: -

7) Growth at 45 ° C: +

8) Indole production: -

9) Lactic acid production: +

10) Fermentation experiments and identification with API 50 CH kit from Biomerieux

0-24 per Whether or not to use Per 25-49 Whether or not to use 0 Control - 25 esculin + 1 glycerol - 26 Living + 2 Erythritol - 27 Cellobios + 3 D arabinose - 28 Maltose + 4 L arabinose - 29 lactose + 5 ribos - 30 Melibiose - 6 D xylose - 31 sucrose + 7 L xylose - 32 Trehalose + 8 Adonitor - 33 inulin - 9 beta methyl-D-xyloside - 34 Melitos - 10 galactose + 35 raffinos - 11 glucose + 36 amidon + 12 fructose + 37 glycogen - 13 Manno + 38 xylitol - 14 sorbos - 39 Gentiobios + 15 Lambos - 40 D Toranos + 16 Dorsitol - 41 License Source - 17 Inositol - 42 D Tagatos + 18 Mannitol - 43 D Fuchos - 19 sorbitol - 44 L Fuchos - 20 [alpha] -methyl-D-mannoside - 45 D arabitol - 21? -Methyl-D-glucoside - 46 L arabitol - 22 N-acetyl-glucosamine + 47 Gluconate - 23 Amigalline + 48 2-keto-gluconate - 24 Arbutin + 49 5-keto-gluconate -

On the other hand, the novel Lactobacillus acidophilus ( Lactobacillus < RTI ID = 0.0 > acidophilus HY7037 as an active ingredient may be used alone or in combination with pharmaceutically acceptable excipients in combination with excipients such as tablets, And the like.

For humans, a typical daily dose may range from 1 to 30 mg / kg body weight, and may be administered in single or divided doses. However, the actual dosage should be determined in light of various relevant factors such as the route of administration, the age, sex and weight of the patient, the health condition, and the severity of the disease.

Of course, the pharmaceutical composition for preventing and / or treating metabolic syndrome containing novel Lactobacillus acidophilus HY7037 having an insulin resistance improving effect of the present invention as an active ingredient has little toxicity and side effects, It is a medicine that can be used safely even when taken for a long time.

Further, the composition containing the novel Lactobacillus acidophilus HY7037 having an insulin resistance improving effect of the present invention as an active ingredient can be used as a food, a food additive, a beverage, a beverage additive, a fermented milk, a health functional food, etc. . When used as a food, a food additive, a beverage, a beverage additive, or a health functional food, it is possible to use various foods, fermented milk, meat, beverage, chocolate, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, But may be, but not limited to, a combination, a mainstream and other health functional food.

In particular, the fermented milk containing the novel Lactobacillus acidophilus HY7037 having an insulin resistance improving effect as an active ingredient can be obtained from Lactobacillus acidophilus HY7037, a lactic acid bacteria culture solution, Mixed juice syrups are homogenized at a constant ratio of 150 bar, cooled to below 10 ° C, and packaged in containers to produce fermented milk.

Also, functional drink containing Lactobacillus ash FIG filler's (Lactobacillus acidophilus) of new HY7037 having an insulin resistance-improving effect as the active ingredient is mixed juice syrup, Lactobacillus ash FIG filler's (Lactobacillus acidophilus HY7037 and water are mixed at a constant ratio and homogenized at 150 bar, cooled to below 10 ° C, and packaged in small containers such as glass bottles and PET bottles to produce functional beverages.

In addition, new Lactobacillus acidophilus ( Lactobacillus < RTI ID = 0.0 > acidophilus HY7037 as an active ingredient contains not only Lactobacillus acidophilus HY7037 but also vitamin B ₁ , B ₂ , B ₅ , B ₆ , E and acetic acid Ester, nicotinic acid amide, oligosaccharide and the like may be added, and other food additives may be added.

As described above, the present invention provides a method for inhibiting insulin sensitivity, comprising the steps of: decreasing IRS-1 (Tyr), increase of AKT protein phosphorylation, increase of GLUT4 gene and protein expression as insulin resistance is induced by intracellular TNF- , A pharmaceutical composition containing Lactobacillus acidophilus HY7037 as an active ingredient, which has an activity of improving insulin resistance according to an increase in final sugar absorption due to an increase in expression of PGC-1 alpha, a fermented milk, a drink, a functional food Can be applied to a product having an effect of preventing and improving a metabolic syndrome.

1 is a graph showing the degree of glucose uptake by L6 skeletal muscle cells induced by insulin resistance of Lactobacillus acidophilus HY7037.
FIG. 2 is a graph showing changes in the amount of GLUT4 gene expression involved in glucose uptake by L6 skeletal muscle cell treatment induced by insulin resistance of Lactobacillus acidophilus HY7037. FIG.
FIG. 3 is a graph showing changes in the expression level of PPARγ gene involved in insulin sensitivity by L6 skeletal muscle cell treatment induced by insulin resistance of Lactobacillus acidophilus HY7037. FIG.
FIG. 4 is a graph showing changes in the expression level of PGC-1 alpha gene involved in insulin sensitivity by L6 skeletal muscle cell treatment induced by insulin resistance of Lactobacillus acidophilus HY7037.
FIG. 5 is a graph showing changes in the amount of IL-6 gene expression, which is a type of inflammatory cytokine induced by L6 skeletal muscle cell treatment induced by insulin resistance of Lactobacillus acidophilus HY7037.
FIG. 6 is a photograph showing changes in p-IRS (Tyr), p-AKT and GLUT4 protein expression by L6 skeletal muscle cell treatment induced by insulin resistance of Lactobacillus acidophilus HY7037.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following embodiments are not intended to limit the scope of the present invention, and ordinary variations by those skilled in the art within the scope of the technical idea of the present invention are possible.

≪ Example 1 >

Lactobacillus acidophilus ( Lactobacillus acidophilus ) Preparation of freeze-dried powder including HY7037

Lactobacillus acidophilus HY7037 2 × 10 ⁹ of the present invention was inoculated into MRS broth and batch cultured at 37 ° C. for 24 hours in an incubator. The supernatant was removed by centrifugation at 4000 rpm for 15 minutes, washed with phosphate buffered saline (PBS), and centrifuged at 4000 rpm for 15 minutes to remove the supernatant. The collected cells were mixed with a skim milk powder as a preservative at a weight ratio of 1: 1 and lyophilized to prepare a powder.

On the other hand, the Lactobacillus asophilus acidophilus HY7037 may be provided in the form of a lyophilized powder or a culture as described above.

≪ Example 2 >

Lactobacillus acidophilus ( Lactobacillus acidophilus ) Preparation of a pharmaceutical composition containing HY7037 as an active ingredient

Manufacture of liquid agent

100 mg of lyophilized powder containing Lactobacillus acidophilus HY7037 of Example 1, 10 g of isomerized sugar, and 5 g of mannitol were dissolved in purified water in accordance with the usual method for preparing a liquid preparation, Was added, and purified water was added to adjust the total volume to 100 ml. The solution was filled in a brown bottle and sterilized to prepare a liquid preparation.

Preparation of capsules

100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate were mixed thoroughly with 100 mg of the lyophilized powder containing Lactobacillus acidophilus HY7037 of Example 1, To prepare hard gelatin capsules.

≪ Example 3 >

Lactobacillus acidophilus ( Lactobacillus acidophilus ) Preparation of fermented milk containing HY7037 as active ingredient

The method for producing fermented milk containing Lactobacillus acidophilus HY7037 as an active ingredient of the present invention is as follows.

The lactic acid bacteria culture solution was prepared by stirring 95.36% by weight of crude oil and 4.6% by weight of skim milk powder (or mixed powdered milk) with a specific gravity of 1.0473 to 1.0475 at 15 ° C, a titratable acidity of 0.200 to 0.220%, a pH of 6.55 to 6.70, Of Brix ⁰ was mixed to be about 16.3 to 16.5%. After mixing, the mixture was heat-treated with UHT (sterilized at 135 ° C. for 2 seconds), cooled to 40 ° C., added with 0.02% by weight of Streptococcus thermophilus and Lactolytic enzyme (Valley laboratory, USA) The total number of lactic acid bacteria in the medium was 1.0 X 10 ⁹ cfu / ml or more, the titratable acidity was 0.89 to 0.91%, and the pH was 4.55 to 4.65. Then, the mixed juice syrup was prepared by mixing 13 wt% of liquid fructose, 5 wt% of white sugar, 10.9 wt% of mixed juice concentrate 56Brix ⁰ , 1.0 wt% of pectin, 0.1 wt% of fresh fruit mix essence and 70 wt% of purified water at 30-35 째 C Mixed with stirring, heat-treated with UHT (sterilized at 135 ° C for 2 seconds), and cooled. Then, 0.15% by weight of the lyophilized powder containing 69.5% by weight of the above-mentioned culture liquid of lactic acid bacteria and Lactobacillus acidophilus HY7037 of Example 1 and 30.4% by weight of the mixed fruit syrup were combined and homogenized at 150 bar, Lt; RTI ID = 0.0 > C < / RTI > to produce Lactobacillus acidophilus HY7037 as an active ingredient.

<Example 4>

Lactobacillus acidophilus ( Lactobacillus acidophilus ) Preparation of vegetable juice containing HY7037 as active ingredient

The method for preparing vegetable juice containing Lactobacillus acidophilus HY7037 of the present invention as an active ingredient is as follows.

Citric acid juice 1 wt.%, Spinach juice 1 wt.%, Lettuce juice 2 wt.%, Celery juice 1 wt.%, Anhydrous citric acid 0.2 wt.%, 0.1% by weight of a lyophilized powder containing Lactobacillus acidophilus HY7037 of Example 1 was uniformly mixed in a blender and sterilized at 85 to 97 ° C using a sterilizer to obtain Lactobacillus acidophilus ( Lactobacillus acidophilus) ) Vegetable juice containing HY7037 as an active ingredient was prepared.

&Lt; Example 5 >

Lactobacillus acidophilus ( Lactobacillus acidophilus ) Preparation of functional beverage containing HY7037 as active ingredient

A method for producing a functional beverage containing Lactobacillus acidophilus HY7037 of the present invention as an active ingredient is as follows.

First, the mixed juice syrup contained 13 wt% of liquid fructose, 2.5 wt% of white sugar, 2.5 wt% of brown sugar, 10.9 wt% of concentrated juice concentrate, 1.0 wt% of pectin, 0.1 wt% of fresh fruit mix essence, and 70 wt% of purified water The mixture was stirred at 30 to 35 ° C, mixed, and heat-treated by UHT (sterilized at 135 ° C for 2 seconds) and cooled.

Then, 30.4% by weight of the mixed fruit juice syrup prepared by the above method was combined with 0.1% by weight of freeze-dried powder containing Lactobacillus acidophilus HY7037 of Example 1 and 69.5% by weight of the remaining purified water, After cooling to below 10 ° C, the product was packaged in a glass bottle, PET bottles, etc., to prepare a functional beverage containing Lactobacillus acidophilus HY7037 as an active ingredient.

&Lt; Example 6 >

Lactobacillus acidophilus ( Lactobacillus acidophilus ) Preparation of Health Functional Foods Containing HY7037 as Active Ingredient

(Vitamin B ₁ , B ₂ , B ₅ , B ₆ , E and acetic acid ester, nicotinic acid amide) were added to 0.1 wt% of the lyophilized powder containing Lactobacillus acidophilus HY7037 of Example ₁ , And oligosaccharide were added in an amount of 10 parts by weight based on 100 parts by weight of the lyophilized powder containing Lactobacillus acidophilus HY7037 of Example 1 and mixed in a high-speed rotary mixer. 10 parts by weight of sterilized purified water was added to the mixture, mixed and molded into granules having a diameter of 1 to 2 mm. The molded granules were dried in a vacuum drier at 40 to 50 DEG C and then passed through 12 to 14 mesh to prepare uniform granules. The granules thus prepared were extruded in suitable amounts to be purified or powdered or filled into hard capsules to prepare hard capsule products.

&Lt; Test Example 1 >

Lactobacillus acidophilus ( Lactobacillus acidophilus ) Influence of HY7037 on blood glucose absorption

As for the utilization of sugar cells in muscle cells, insulin secreted from pancreatic β-cells and insulin receptor in muscle cells, GLUT4 (glucose transporter type 4), are closely related. Sugars such as glucose in the blood are used in various organs such as adipocytes and muscle tissue. In the case of muscle cells, insulin action causes insulin signal due to the action of insulin receptor Operation. Finally, the expression of GLUT4 and the increase in translocation in muscle cells allows glucose in the blood to enter the muscle, allowing it to be synthesized and used with glycogen and the like.

However, when fatty acids such as TNF-α and palmitic acid act in this process, an abnormality in the insulin signaling mechanism results in an abnormal expression of GLUT4 and translocation, resulting in poor blood glucose inflow .

Therefore, in order to ascertain the increase in blood glucose uptake in the case of Lactobacillus acidophilus HY7037 of the present invention in which the intracellular TNF-a of the Lactobacillus acidophilus HY7037 is decreased due to the decrease of blood glucose absorption, glucose uptake assay (glucose uptake assay) (glucose uptake).

1-1. Muscle cell culture

L6 cell myoblast, a skeletal muscle cell derived from rat, was purchased from the Korean Cell Line Bank (cellbank.snu.ac.kr) and used for the animal cells used in this test. Cell culture was carried out in a medium containing 90% DMEM medium (Gibco #) and 10% FBS (Gibco # 16000) for maintenance of normal cells, and subculture was performed twice a week at a ratio of 1: 8.

1-2. Securing cell extract of lactic acid bacteria

Lactobacillus acidophilus HY7037 of the present invention was cultured in MRS medium (Difco) at 37 占 폚 for 24 hours. The cells were centrifuged, washed with PBS 2-3 times, and the remaining medium was removed. The cells were lyophilized and pulverized, and then the number of bacteria was measured by serial dilution. The cells were resuspended in PBS to a concentration of 10 ¹⁰ cfu / ml and sonicated to lysis the cells. After centrifugation, the supernatant was used for the test as a lactic acid bacterium cell extract.

1-3. Differentiation of muscle cells

The L6 skeletal muscle cells were subcultured in a 96-well plate at 5 × 10 ⁵ cells / well and cultured for 3 to 4 days using DMEM supplemented with 2% FBS.

1-4. Lactobacillus ash FIG filler's (Lactobacillus acidophilus) glucose absorption measurements in the L6 skeletal muscle cells, insulin resistance induced by TNF-α by the processing strain HY7037

Lactobacillus acidophilus HY7037 of the present invention was pre-treated at ¹⁰⁶ cfu / well for 24 hours in the differentiated L6 skeletal muscle cells of Test Example 1-3, and the following day TNF-α (100 ng / ml ) For 6 hours at a rate of 200 쨉 l per well to induce insulin resistance.

After removing the culture medium, 2-NBDG (1:70), insulin (500 μM) treated with the fluorescent substance, glucose-containing lactose as the lactobacillus ash of Test Example 1-2, Lactobacillus acidophilus HY7037 extract was mixed together and treated with 100 μl each for 30 minutes.

200 [mu] l of each well was washed with an assay buffer in a kit of Cayman Chemical Co. for 5 minutes, and then 100 [mu] l of assay buffer was added to the same plate. Using a plate reader, Excitation / emission = 485/535 nm fluorescence measurement.

The results are shown in Fig.

&Quot; Negative control group &quot; represents a group in which differentiated L6 skeletal muscle cells were not treated with both insulin and TNF-alpha and Lactobacillus acidophilus HY7037 of the present invention,

The 'positive control group' represents the group treated with only insulin (500 μM) to differentiated L6 skeletal muscle cells,

'Experimental group 1' represents the group treated with only insulin (500 μM) and TNF-α (100 ng / ml) in differentiated L6 skeletal muscle cells,

&Quot; Experimental group 2 &quot; represents a group treated with insulin (500 μM), TNF-α (100 ng / ml) and Lactobacillus acidophilus HY7037 of the present invention to differentiated L6 skeletal muscle cells.

As can be seen in FIG. 1, glucose uptake by insulin was increased in normal control muscle cells, whereas in experimental group 1, blood glucose uptake was decreased (p <0.01) due to TNF-α in normal muscle cells. However, as in Experiment 2, decreased blood glucose uptake due to TNF- [alpha] was found to be increased (p < 0.05) by treating Lactobacillus acidophilus HY7037 of the present invention.

Therefore, it was confirmed that Lactobacillus acidophilus HY7037 of the present invention increases blood glucose uptake in L6 skeletal muscle cells reduced by TNF-α.

&Lt; Test Example 2 &

Lactobacillus acidophilus ( Lactobacillus acidophilus ) Measurement of expression of GLUT4, PPARγ and PGC-1α in L6 skeletal muscle cells by treatment with HY7037

Myocardial glucose uptake is caused by a protein called GLUT4. In particular, PPARγ (peroxisome proliferator-activated receptor gamma) and PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha) are related to insulin sensitivity and are expressed by insulin. It affects the translocation. Therefore, it was confirmed that the increase of glucose uptake due to the increase of gene expression of GLUT4, PPARγ and PGC-1α of Lactobacillus acidophilus HY7037 of the present invention in L6 skeletal muscle cells induced by insulin resistance by TNF- The expression levels of GLUT4, PPARγ and PGC-1α genes were measured.

2-1. Differentiation of muscle cells

L6 skeletal muscle cells of Test Example 1-1 were subcultured on a 6-well plate at 1 × 10 ⁶ cells / well and cultured for 3 to 4 days using DMEM medium containing 2% FBS.

2-2. Lactobacillus ash FIG filler's (Lactobacillus acidophilus) GLUT4 gene expression level measured in the L6 skeletal muscle cells, insulin resistance induced by TNF-α by the processing HY7037

Lactobacillus acidophilus HY7037 of the present invention was pretreated with 10 ⁷ cfu / well for 24 hours, and the following day, TNF-α (100 ng / ml) was added to the differentiated muscle cells of Test Example 2-1. And treated with 2 ml per well for 6 hours to induce insulin resistance.

After the culture medium was removed, insulin (500 μM) and Lactobacillus acidophilus HY7037 extract of Test Example 1-2 were mixed together and treated in 2 ml portions for 30 minutes.

RNA from muscle cells was obtained from Qiagen's RNA prep. Kit (RNeasy mini kit, Qiagen 74106). CDNA was synthesized from the separated RNA using Quantit reverse transcription kit (Qiagen, Valencia, Calif.). RNA expression was quantitated using Taqman probe and Real-time PCR (applied biosystems, 7500 real time PCR) shown in Table 2 below. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was used as an internal control.

gene Taqman probe Rat GAPDH Rn01775763_g1 Rat solute carrier family 2 (facilitated glucose transporter), member 4, Glut-4 Rn00562597_m1 Rat PPARγ Rn00440945_m1 Rat peroxisome proliferative activated receptor, gamma, coactivator 1 alpha Rn00580241_m1

The results are shown in Fig.

&Quot; Negative control group &quot; represents a group in which differentiated L6 skeletal muscle cells were not treated with both insulin and TNF-alpha and Lactobacillus acidophilus HY7037 of the present invention,

The 'positive control group' represents the group treated with only insulin (500 μM) to differentiated L6 skeletal muscle cells,

'Experimental group 1' represents the group treated with only insulin (500 μM) and TNF-α (100 ng / ml) in differentiated L6 skeletal muscle cells,

&Quot; Experimental group 2 &quot; represents a group treated with insulin (500 μM), TNF-α (100 ng / ml) and Lactobacillus acidophilus HY7037 of the present invention to differentiated L6 skeletal muscle cells.

As can be seen in FIG. 2, GLUT4 gene expression, which is involved in glucose uptake by insulin, is increased in the normal muscle cells of the positive control group, whereas in the test group 1, TNF- GLUT4 gene expression was reduced. However, as shown in Experiment 2, the gene expression of GLUT4 reduced due to TNF-α increased the expression of GLUT4 gene by treating Lactobacillus acidophilus HY7037 of the present invention. Therefore, it was confirmed that Lactobacillus acidophilus HY7037 of the present invention increases the amount of GLUT4 expression reduced by TNF-α, thereby increasing blood glucose uptake.

2-3. Lactobacillus ash FIG filler's (Lactobacillus acidophilus) PPARγ gene expression level measured in the L6 skeletal muscle cells, insulin resistance induced by TNF-α by the processing HY7037

The expression level of the PPAR gamma gene was measured by the same test method as in Test Example 2-2.

The results are shown in Fig.

&Quot; Negative control group &quot; represents a group in which differentiated L6 skeletal muscle cells were not treated with both insulin and TNF-alpha and Lactobacillus acidophilus HY7037 of the present invention,

The 'positive control group' represents the group treated with only insulin (500 μM) to differentiated L6 skeletal muscle cells,

'Experimental group 1' represents the group treated with only insulin (500 μM) and TNF-α (100 ng / ml) in differentiated L6 skeletal muscle cells,

&Quot; Experimental group 2 &quot; represents a group treated with insulin (500 μM), TNF-α (100 ng / ml) and Lactobacillus acidophilus HY7037 of the present invention to differentiated L6 skeletal muscle cells.

As can be seen in FIG. 3, in the normal control muscle group, the expression of PPARγ gene, which is involved in insulin sensitivity and glucose uptake, was increased by insulin. In contrast, in experimental group 1, insulin sensitivity and glucose uptake due to TNF- PPARγ gene expression involved in glucose uptake was reduced. However, as in experimental group 2, the expression of PPAR gamma, which is involved in decreased insulin sensitivity and glucose uptake due to TNF-α, increases the expression of PPARγ gene by treating Lactobacillus acidophilus HY7037 of the present invention Could know. Therefore, it was confirmed that Lactobacillus acidophilus HY7037 of the present invention increases the amount of PPARγ expressed by TNF-α, thereby increasing blood glucose absorption.

2-4. Lactobacillus ash FIG filler's (Lactobacillus acidophilus) PGC-1α gene expression level measured in the L6 skeletal muscle cells, insulin resistance induced by TNF-α by the processing HY7037

The expression level of PGC-1 alpha gene was measured by the same test method as in Test Example 2-2.

The results are shown in Fig.

&Quot; Negative control group &quot; represents a group in which differentiated L6 skeletal muscle cells were not treated with both insulin and TNF-alpha and Lactobacillus acidophilus HY7037 of the present invention,

The 'positive control group' represents the group treated with only insulin (500 μM) to differentiated L6 skeletal muscle cells,

'Experimental group 1' represents the group treated with only insulin (500 μM) and TNF-α (100 ng / ml) in differentiated L6 skeletal muscle cells,

&Quot; Experimental group 2 &quot; represents a group treated with insulin (500 μM), TNF-α (100 ng / ml) and Lactobacillus acidophilus HY7037 of the present invention to differentiated L6 skeletal muscle cells.

As can be seen from FIG. 4, in the normal muscle cells as positive control group, expression of PGC-1 alpha gene which is involved in insulin sensitivity and glucose uptake was increased by insulin, whereas in the test group 1, Sensitivity and PGC-1α gene expression involved in glucose absorption were reduced. However, as in Experiment 2, the expression of PGC-1 alpha gene, which is involved in insulin sensitivity and glucose uptake due to TNF-α, is inhibited by treating Lactobacillus acidophilus HY7037 of the present invention, Expression was increased. Therefore, it was confirmed that Lactobacillus acidophilus HY7037 of the present invention increases the expression of PGC-1α reduced by TNF-α, thereby increasing blood glucose absorption.

&Lt; Test Example 3 >

Lactobacillus acidophilus ( Lactobacillus acidophilus ) Measurement of IL-6 expression level in L6 skeletal muscle cells by treatment with HY7037

When insulin-resistant environment is induced in muscle cells, the phosphorylation process of IRS-1 (insulin receptor substrate 1 (Ser)) is increased and mitogen-activated protein kinase (MAPK) and NF-κB Expression of the same inflammatory factors is also increased. This leads to increased expression of IL-6, a type of inflammatory cytokine.

Therefore, in order to confirm the inhibitory effect of the Lactobacillus acidophilus HY7037 treatment of the present invention on the expression of inflammatory cytokines, gene expression of IL-6 was analyzed.

Lactobacillus acidophilus HY7037 was pretreated with 10 ⁷ cfu / well for 24 hours and then incubated with TNF-α (100 ng / ml) for 24 hours in the differentiated L6 skeletal muscle cells of Test Example 2-1 The insulin resistance was induced by treatment with 2 ml per 6 hours.

After the culture medium was removed, insulin (500 μM) and Lactobacillus acidophilus HY7037 extract of Test Example 1-2 were mixed together and treated in 2 ml portions for 30 minutes.

RNA from muscle cells was obtained from Qiagen's RNA prep. Kit (RNeasy mini kit, Qiagen 74106). CDNA was synthesized from the separated RNA using Quantit reverse transcription kit (Qiagen, Valencia, Calif.). RNA expression was quantitated using the Taqman probe and Real-time PCR (applied biosystems, 7500 real time PCR) of Table 3 below. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was used as an internal control.

gene Taqman probe Rat IL-6 Rn01410330_m1

The results are shown in Fig.

&Quot; Negative control group &quot; represents a group in which differentiated L6 skeletal muscle cells were not treated with both insulin and TNF-alpha and Lactobacillus acidophilus HY7037 of the present invention,

The 'positive control group' represents the group treated with only insulin (500 μM) to differentiated L6 skeletal muscle cells,

'Experimental group 1' represents the group treated with only insulin (500 μM) and TNF-α (100 ng / ml) in differentiated L6 skeletal muscle cells,

&Quot; Experimental group 2 &quot; represents a group treated with insulin (500 μM), TNF-α (100 ng / ml) and Lactobacillus acidophilus HY7037 of the present invention to differentiated L6 skeletal muscle cells.

As can be seen from FIG. 5, in the normal control muscle cells, the expression of IL-6 gene related to inflammation did not differ from the negative control group, whereas in the experimental group 1, the TNF- Induction of the environment increased IL-6 gene expression associated with inflammation. However, IL-6 gene expression associated with increased inflammation due to TNF-α, as in Experiment 2, is inhibited by IL-6 gene expression by treating Lactobacillus acidophilus HY7037 of the present invention Could know. Therefore, Lactobacillus acidophilus HY7037 of the present invention can inhibit the expression of IL-6 and improve the inflammatory environment according to insulin resistance in L6 skeletal muscle cells.

<Test Example 4>

Lactobacillus acidophilus ( Lactobacillus acidophilus ) Measurement of IRS-1 (Tyr), AKT phosphorylation and protein expression of GLUT4 in L6 skeletal muscle cells treated with HY7037

Treatment of L6 skeletal muscle cells with TNF-α induces insulin resistance, which increases phosphorylation of IRS-1 (Insulin receptor substrate 1; Ser) and inhibits phosphorylation of IRS-1 (Insulin receptor substrate 1; Tyr) Therefore, it affects the lower signaling pathway of the insulin signal pathway, and ultimately affects the expression of AKT (Protein Kinase B) and GLUT4, which are related to intramuscular blood glucose uptake.

Therefore, the protein expression of GLUT4 is increased by the increase of the protein phosphorylation of IRS-1 (Tyr) and AKT (Protein Kinase B) of Lactobacillus acidophilus HY7037 of the present invention and the improvement of insulin resistance The expression of p-IRS-1 (Tyr), p-AKT and GLUT4 was analyzed to confirm the effect.

That is, the differentiated L6 skeletal muscle cells of Test Example 2-1 were pretreated with Lactobacillus acidophilus HY7037 at 10 ⁷ cfu / well for 24 hours, and the following day, TNF-α (100 ng / ml) And treated with 2 ml per well for 6 hours to induce insulin resistance.

After the culture medium was removed, insulin (500 μM) and Lactobacillus acidophilus HY7037 extract of Test Example 1-2 were mixed together and treated in 2 ml portions for 30 minutes.

The cells were lysed with RIPA buffer and denatured by boiling at 95 ° C for 15 minutes with SDS loading buffer. Proteins were separated by electrophoresis on 8-12% SDS-PAGE gels at a concentration of approximately 40 μg protein and transferred to nitrocellulose membrane. (Santa Cruz, sc-560), AKT (Cell signaling, # 9272), and p-AKT (Cell signaling, # 9271) after blocking for 1 hour and 30 minutes in 5% nonfat milk. Primary antibody (1: 1), such as GLUT4 (Santa Cruz, sc-1607), β-actin (Santa Cruz, sc-47778), p- 500 ~ 1: 1000)] was left at 4 ° C for 24 hours. After washing the membrane with TBS-T 6 times for 5 minutes, the appropriate second antibody was treated at room temperature for 2 hours, and then re-washed with TBS-T. The X- ray film to confirm the result.

The results are shown in Fig.

&Quot; Lane 1 &quot; represents a group in which insulin and TNF-alpha and Lactobacillus acidophilus HY7037 of the present invention are not treated in the differentiated L6 skeletal muscle cells,

'Lane 2' represents the group treated with only insulin (500 μM) to differentiated L6 skeletal muscle cells,

'Lane 3' represents the group treated with only insulin (500 μM) and TNF-α (100 ng / ml) in differentiated L6 skeletal muscle cells,

'Lane 4' represents a group treated with insulin (500 μM), TNF-α (100 ng / ml) and Lactobacillus acidophilus HY7037 of the present invention to differentiated L6 skeletal muscle cells .

'p-IRS1' represents phosphorylated IRS1 and 'p-AKT' represents phosphorylated AKT.

&Quot; beta-actin &quot; indicates experimentation with the same amount of cells.

As shown in FIG. 6, in the normal muscle cells of lane 2, the phosphorylation process of IRS-1 (Tyr) in the insulin signal pathway is increased by insulin, and the increase of p-IRS- GLUT4, which is involved in protein phosphorylation (p-AKT) and glucose uptake, also increases protein expression level compared to Lane 1, whereas when insulin resistance is induced by TNF-α in normal muscle cells of lane 3, (Tyr) phosphorylation of the IRS-1 (Tyr) was suppressed, and the protein expression of the phosphorylation process of the AKT was reduced, and the protein expression of GLUT4, which is involved in glucose uptake, was finally reduced. However, as in lane 4, protein phosphorylation of IRS-1 (Tyr) reduced by TNF-α in normal muscle cells and phosphorylation of its lower signaling mechanism, AKT, and protein expression of GLUT4 were found in Lactobacillus acidophilus ) HY7037, the protein expression level was increased compared to Lane 3. Thus, the Lactobacillus acidophilus HY7037 of the present invention increases the expression of p-IRS (Tyr), p-AKT, and GLUT4, which are reduced by TNF-α, .

Korea Biotechnology Research Institute KCTC12409BP 20130509

Claims (6)

Lactobacillus acidophilus ( Lactobacillus acidophilus), which has the effect of improving insulin resistance by increasing glucose uptake in muscle cells, acidophilus HY7037 (accession number: KCTC 12409BP).
The method according to claim 1,
The intracellular glucose uptake increase is achieved by increasing the expression of IRS-1 (Insulin receptor substrate 1 (Tyr), AKT (Protein Kinase B) and GLUT4 (glucose transporter type 4) Lactobacillus acidophilus HY7037 (accession number: KCTC 12409BP) having an insulin resistance improving effect.
The method according to claim 1,
Wherein the increase in glucose uptake in muscle cells is achieved by increasing expression of PPAR gamma (peroxisome proliferator-activated receptor gamma) and PGC-1 alpha (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) Lactobacillus acidophilus HY7037 (accession number: KCTC 12409BP) having resistance-improving efficacy.
A method for preventing and / or preventing metabolic syndrome through improvement of insulin resistance characterized by containing Lactobacillus acidophilus HY7037 (Accession No: KCTC 12409BP) of any one of claims 1 to 3 as an active ingredient A pharmaceutical composition for therapeutic use.
A food composition for preventing metabolic syndrome through improvement of insulin resistance characterized by comprising Lactobacillus acidophilus HY7037 (accession number: KCTC 12409BP) of any one of claims 1 to 3 as an active ingredient .
6. The method of claim 5,
Wherein the food composition is vegetable juice, functional beverage, health functional food, fermented milk.

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