JP2013203707A - Anti-obesity agent consisting of 1,5-d-anhydro fructose - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an activity inhibitor of glycerol-3-phosphate dehydrogenase which has high safety, is easily obtained, and is able to be used for a human being, and to provide a production inhibiting agent of a transcription factor PPARγ and C/EBPβ.SOLUTION: An activity inhibitor of glycerol-3-phosphate dehydrogenase composed of 1,5-D-anhydro fructose, and a production inhibiting agent of a lipogenesis transcription factor PPARγ and/or C/EBPβ are disclosed.

Description

  The present invention relates to an anti-obesity agent comprising 1,5-D-anhydrofructose. More specifically, the present invention relates to an activity inhibitor or production inhibitor for suppressing adipocyte hypertrophy by 1,5-D-anhydrofructose.

In recent years, it has become a global social problem that the number of obese people who develop as a result of overeating or lack of exercise exceeds the consumed energy is increasing year by year. Obesity develops hypertension, abnormal glucose metabolism (diabetes) and abnormal lipid metabolism (dyslipidemia) due to abnormal secretion of various adipocytokines secreted from fat cells. When obesity is a basic disease, two or more of hypertension, diabetes, and dyslipidemia are accompanied by metabolic syndrome. It is known that metabolic syndrome is an onset of atherosclerosis and further leads to serious diseases such as angina pectoris, myocardial infarction, and cerebral infarction.
Glycerol-3-phosphate dehydrogenase is an enzyme related to synthesis of triacylglycerol from glucose, and is an enzyme that converts dihydroxyacetone phosphate, which is an intermediate pathway thereof, to glycerol-3-phosphate. Inhibition of this enzyme inhibits the synthesis of glycerol-3-phosphate among acyl CoA and glycerol-3-phosphate, which are two precursors of triacyl glycerol, resulting in triacylglycerol Synthesis is inhibited. Therefore, inhibition of this enzyme is thought to lead to anti-obesity, and this glycerol-3-phosphate dehydrogenase inhibitor has attracted attention in the fields of medicine and food.
As this inhibitor, α-lipoic acid, flavonoids, phenolic acids, gymnemic acid and the like have been reported.
In addition, PPARγ and C / EBPβ are responsible for the transcriptional control of many genes that bring about the characteristics of adipocytes such as fat synthesis and fatty acid uptake. When the expression of these transcription factors is suppressed, differentiation from preadipocytes to adipocytes and hypertrophy of adipocytes are suppressed.
When the expression of PPARγ and C / EBPβ, which are transcription factors for adipocyte differentiation, is inhibited, adipogenic enzyme activity is lowered and adipocyte differentiation is inhibited.
As countermeasures for obesity, it is effective to reduce intake energy by reducing the amount of meals and increase energy consumption by increasing exercise amount. However, it is tired and it is difficult to carry out these activities continuously at the present time when transportation and transportation methods are developed. Therefore, there is an increasing expectation for weight control by ingesting a food having anti-obesity action, a food for specified health use, a pharmaceutical product, a quasi-drug, etc. as a means for obtaining an anti-obesity effect more easily.
Substances exhibiting anti-obesity action include inhibiting the activity of neutral fat synthesis, glycerol-3-phosphate dehydrogenase activity or transcription factors PPARγ and C / EBPβ involved in adipocyte differentiation. Conceivable.
1,5-D-anhydrofructose (hereinafter referred to as 1,5-AF) is a monosaccharide produced by degrading starch, glycogen, etc. with the enzyme α-1,4-glucan lyase, and has antioxidant and antibacterial properties. It has also been reported that it has anti-inflammatory properties, anti-allergic properties and the like as physiological effects (Patent Documents 1 to 4). A patent application has also been filed as an adiponectin production enhancer (Patent Document 5). 1,5-AF is used as a food material, and is a material that has no problem in terms of safety as a substance to be taken orally.

Japanese National Patent Publication No. 9-505988 JP 2001-89377 A JP 2006-306814 A JP 2007-091644 A JP 2008-195630 A

An object of the present invention is to provide an activity inhibitor of glycerol-3-phosphate dehydrogenase, a transcription factor PPARγ and a production inhibitor of C / EBPβ, which are highly safe, easily available, and usable for humans. is there.
Other objects and advantages of the present invention will become apparent from the following description.

As a result of intensive studies to solve the above problems, the present inventors have found that 1,5-AF inhibits the activity of glycerol-3-phosphate dehydrogenase on adipocytes, and further relates to differentiation of adipocytes. The inventors have found that the production of transcription factors PPARγ and C / EBPβ is suppressed, and have completed the present invention.
That is, according to the present invention, the above objects and advantages of the present invention are as follows.
It is achieved by an activity inhibitor of glycerol-3-phosphate dehydrogenase consisting of 1,5-D-anhydrofructose.
According to the present invention, the above objects and advantages of the present invention are secondly achieved by a production inhibitor of adipose synthesis transcription factor PPARγ and / or C / EBPβ comprising 1,5-D-anhydrofructose. .

  1,5-AF, which is an active ingredient of the present invention, is safe for humans and the environment. By administering 1,5-AF to humans, lipid metabolism and sugar metabolism can be activated, so obesity, diabetes, It is extremely useful for the prevention and improvement of lifestyle-related diseases such as insulin resistance, hyperglycemia, dyslipidemia (dyslipidemia), arteriosclerosis, and hypercholesterolemia.

Relationship between preadipocyte viability and 1,5-AF concentration. The optical microscope photograph which shows the relationship between the amount of TG accumulation | storage of a preadipocyte, and the density | concentration of 1, 5- AF. Relationship between GPDH activity of preadipocytes and the concentration of 1,5-AF. The relationship between the expression level of PPARγ in preadipocytes and the concentration of 1,5-AF. Relationship between the expression level of C / EBPβ in preadipocytes and the concentration of 1,5-AF. Relationship between viability of mature adipocytes and concentration of 1,5-AF. Relationship between the amount of TG accumulated in mature adipocytes and the concentration of 1,5-AF. The optical micrograph which shows the relationship between the size of the fat droplet of a mature fat cell, and the density | concentration of 1, 5- AF. Relationship between the amount of PPARγ expression in mature adipocytes and the concentration of 1,5-AF.

1,5-AF used in the present invention can be prepared by a known method, for example, the method described in Patent Document 1.
That is, 1,5-AF can be obtained by allowing the enzyme α-1,4-glucan lyase extracted from red alga ogonori to act on starch.
In the present invention, 1,5-AF does not act as a cytotoxin on adipocytes, and acts as the activity inhibitor and the production inhibitor even if the adipocytes are either preadipocytes or mature adipocytes. Is expressed.
In the present invention, 1,5-AF (1,5-anhydro-D-fructose) may be in any form and is not particularly limited. It can also take the form of a dilute aqueous solution, a highly concentrated aqueous solution, a lyophilized powder, a crystalline state or the like. Other components may be present in addition to 1,5-AF. It may be in a form mixed with other components such as sugars such as glucose and maltodextrin, ethyl alcohol, proteins, amino acids, lipids, dietary fiber, vitamins, minerals and the like.
In addition to 1,5-AF, the agent of the present invention includes other glycerol-3-phosphate dehydrogenase activity inhibitory compounds, transcription factor PPARγ and C / EBPβ production inhibitory compounds as long as the effects of the present invention are exhibited. It is also possible.

The glycerol-3-phosphate dehydrogenase activity inhibitor, the transcription factor PPARγ and the production inhibitor of C / EBPβ of the present invention can be administered according to their dosage forms by various methods known per se. The amount, administration site, administration interval, period, etc. can be determined in consideration of the age and weight of the patient, the medical condition, or other drugs and treatment methods. The administration method can be, for example, oral administration, direct injection into the body such as intravenous, subcutaneous, intraperitoneal, or external use by injection or infusion, and is not particularly limited.
The dose of the agent in the present invention varies depending on the dosage form, administration method, or symptoms to be prevented or treated. For example, the dose per kg of body weight is 0.1 μg to 1,000 mg as 1,5-AF. However, it can be preferably 1 mg to 1,000 mg, and can be administered at an appropriate dosing frequency such as once or several times a day or once every several days.

  Examples of the form of the glycerol-3-phosphate dehydrogenase activity inhibitor and the production inhibitor of transcription factors PPARγ and C / EBPβ of the present invention include tablets, capsules, granules, powders, syrups, injections, and suppositories. Inhalants and the like can be mentioned, but are not particularly limited. In addition, ingredients necessary for preparing a preparation, such as preparation carriers and excipients, binders, extenders, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, flavoring agents , Fragrances, coating agents, carriers, stabilizers and the like.

Furthermore, as long as the effect of the present invention is exhibited, it is possible to include other anti-atherosclerotic drugs, anti-diabetic drugs, or other pharmacological or nutritional components.
In addition, the use of the glycerol-3-phosphate dehydrogenase activity inhibitor of the present invention and the production inhibitor of transcription factors PPARγ and C / EBPβ is not limited to pharmaceutical use, but is incorporated into quasi drugs, foods, beverages, etc. It is also possible to do. For example, 1,5-AF can be added to food to take the form of a functional food intended for the treatment of arteriosclerosis.
As a form in the case of using this invention as a foodstuff, various foods, such as a drink, dairy products, breads, cakes, noodles, confectionery, frozen food, are mentioned.

  The glycerol-3-phosphate dehydrogenase activity inhibitor and transcription factor PPARγ or C / EBPβ production inhibitor of the present invention can also be administered to mammals other than humans. That is, in that case, obesity, hypertension, dyslipidemia (dyslipidemia) by administering appropriate amounts of glycerol-3-phosphate dehydrogenase activity inhibitor and transcription factor PPARγ or C / EBPβ production to mammals. ), Prevention and treatment of diabetes, arteriosclerosis, liver fibrosis, and cancer.

Examples of the present invention are shown below, but the present invention is not limited to these examples.
1. Experimental method (1) Culture of preadipocytes Mouse fibroblast 3T3-L1 cells are 10% fetal bovine serum (FBS), penicillin (50 units / ml, Meiji Seika) and streptomycin (50 mg / ml, Meiji Seika Co., Ltd.) )) In a Dulbecco's modified Eagle's medium (DMEM) medium (Nissui Pharmaceutical Co., Ltd.) and pre-cultured for 6 to 7 days in an incubator adjusted to 37 ° C. and 5% CO ₂ . After the cells were detached by treatment with trypsin / EDTA solution, the number of cells was adjusted to 1.0 × 10 ⁵ cells / ml.
(2) Passage of preadipocytes After removing the medium and washing the cells with phosphate buffer (PBS), the cells were allowed to act with trypsin / EDTA solution until some cells were detached, and then neutralized solution (FBS: PBS = 1: 2) was added. After collecting the cells, they are suspended and centrifuged (3,000 rpm, 5 minutes, 4 ° C.), the supernatant is removed, DMEM medium is added, and the number of cells is 1.0 × 10 ^{5 to} 1.0 × 10 ^6. It adjusted so that it might become cells / ml.
(3) Differentiation induction from preadipocytes to mature adipocytes After culturing the preadipocytes to a preconfluent state, bovine-derived insulin (0.2 μM, Wako Pure Chemical Industries, Ltd.), dexamethasone 0.25 μM, sum Koganei Pharmaceutical Co., Ltd.) and DMEM medium supplemented with 3-isobutyl-1-methylxanthine (0.5 mM, Wako Pure Chemical Industries) were replaced. Two days later, the medium was replaced with a medium supplemented with only insulin (0.2 μM), and thereafter, the medium was replaced with a new DMEM medium every two days. The sample was added every 2 days when the medium was changed.

(4) 3T3-L1 matured adipocyte culture 1,5-AF was added to 3T3-L1 matured adipocytes where differentiation induction was completed and lipid droplets were accumulated, and various measurements were performed after 3 days of culture. It was.
(5) Measurement of preadipocyte survival rate (Neutral red method)
Neutral red passes through intact cell membranes and is taken up by lysosomes in viable cells. Utilizing this property, the amount of Neutral red incorporated into the cells was measured, and the cell viability was calculated by measuring the proportion of viable cells.
24 hours after adding 1,5-AF, the medium was removed, and Neutral red reagent (0.25 mg / ml, Wako Pure Chemical Industries, Ltd.) was added to the cells to a final concentration of 50 μg / ml. It was. After incubation at 37 ° C for 2 hours, the cells were washed twice with a mixture of 1% formaldehyde, 1% calcium chloride, and 98% ultrapure water, and then continuously with 1% acetic acid, 50% ethanol, and 49% ultrapure water. Containing 1 ml of decolorized extract was added to the cells and allowed to stand for 30 minutes.
The amount of Neutral red absorption of lysosomes was measured at 540 nm using an absorptiometer (Beckman Coulter, DU530). The cell viability was expressed as a relative value when the absorbance of the control was 100%.

(6) Amount of triacylglycerol (TG) accumulated (Oil red O staining method)
The oil red O / isopropanol saturated solution was shaken well, mixed at a ratio of saturated solution: ultra pure water = 6: 4, and then filtered using a 0.45 μm filter after 10 minutes. After completion of induction of cell differentiation, the medium was removed by suction with an aspirator and washed twice with PBS.
After fixing with 70% ethanol for 30 seconds, 2 ml of Oil red O staining solution was added and left at room temperature for 2 hours. After washing with 50% ethanol for 10 seconds, it was washed with deionized water until the redness of the water disappeared. Observation was performed with an inverted microscope (OLYMPUS IX-70) at a magnification of 200 in a bright field, and after photography, 3 ml of isopropanol was added to extract Oil red O staining solution. Further, the absorbance of the extract was measured at a wavelength of 520 nm.

(7) Measurement of glycerol-3-phosphate dehydrogenase (GPDH) activity A 100 mM triethanolamine / 2.5 mM EDTA solution was kept at 28 ° C. in a test tube on a water bath. After completion of the main culture, the dish was washed twice with PBS, 300 μl of a 100 mM triethanolamine / 2.5 mM EDTA solution was added, and the cells were recovered with a cell scraper. Thereafter, the cells were disrupted by sonication, rapidly centrifuged (13,000 rpm, 5 minutes, 4 ° C.), and the supernatant was used as a cell solution. To a test tube on the water bath, add 6 mM 2-mercaptoethanol (final concentration 0.1 mM), 7.2 mM NADH (final concentration 0.12 mM), 12 mM dihydroxyacetone phosphate (final concentration 0.2 mM), cell solution, Measurement was performed at an absorbance of 340 nm for 3 minutes. The activity of GPDH was calculated using the NADH extinction coefficient of 6.22 mM ⁻¹ cm ⁻¹ and calculated based on the change in NADH per minute. Enzyme activity was expressed as a value relative to the control (100%).

(8) Detection of transcription regulatory factor expression (Western blotting method)
48 hours after the addition of 1,5-AF, the dish was washed once with PBS, the cells were collected with Buffer X (Table 1), and centrifuged (10,000 rpm, 10 minutes, 4 ° C.). Thereafter, the supernatant was removed and Buffer Y (Table 2) was added. Then, freeze-thaw was performed twice and the cell membrane was destroyed by ultrasonic treatment. Centrifugation was quickly performed (3,500 rpm, 10 minutes, 4 ° C.) to obtain a precipitate containing a nuclear fraction.
Buffer Y was added to the precipitate, and pipetting was performed well to disperse the precipitate, followed by cooling (30 minutes, 4 ° C.). In addition, the nuclei were destroyed by sonication. Then, it centrifuged quickly (15,000 rpm, 10 minutes, 4 degreeC), and used the supernatant as a nucleoprotein. 20 μg of protein was separated by SDS-polyacrylamide gel electrophoresis (PAGE) using the method of Laemmli et al. The gel from which the protein was separated by SDS-PAGE was blotted using BIO-RAD Trans blot SD. After completion of the blotting, the PVDF membrane was subjected to detection of transcription regulatory factors by the enzyme antibody method using SNAPid (Milipore). The antibodies used were anti-PPARγ mouse monoclonal antibody (Santa Cruz Biotechnology), anti-C / EBPα rabbit polyclonal antibody (Santa Cruz Biotechnology), and anti-C / EBPβ mouse monoclonal antibody (Santa Cruz Biotechnology). In addition, β-actin and tubulin β, which are known to be constantly expressed, were used as controls.

2. Experimental Results The results of examining the effect of 1,5-AF on the preadipocyte survival rate are shown in FIG. The cell viability was not different from that of the control group when the 1,5-AF concentration in the medium was in the range of 200 to 800 μg / ml, and thus 1,5-AF did not show cytotoxicity against preadipocytes.
When preadipocytes differentiate into mature adipocytes, lipid droplets accumulate in the cells. In order to evaluate the degree of differentiation from preadipocytes to mature adipocytes, lipid droplets were stained by the Oil red O method. As a result, as shown in FIG. 2, in the lipid fat droplets of the preadipocytes stained by the Oil red O method, many fat droplets of various sizes are observed in the control. When AF was added, intracellular lipid droplets were reduced in size and number. Furthermore, when 800 μg / ml 1,5-AF was added, almost no lipid droplets were observed.
From this, it was found that the addition of 1,5-AF suppresses differentiation of preadipocytes into adipocytes.

TG is biosynthesized from fatty acid and glucose, but GPDH is also a rate-limiting enzyme for TG synthesis because GPDH is also involved in the synthesis stage. Therefore, GPDH activity was measured. As a result, GPDH activity was inhibited depending on the addition concentration of 1,5-AF in the range of 200 to 800 μg / ml (FIG. 3).
PPARγ and C / EBPα are nuclear transcriptional regulators and play an important role in adipocyte differentiation. C / EBPβ is located upstream of PPARγ and C / EBPα. Therefore, the effect of 1,5-AF on intracellular PPARγ and C / EBPβ levels was examined. As a result, as shown in FIGS. 4 and 5, 1,5-AF suppressed the expression of these two transcription factors in a concentration-dependent manner. From this, it was considered that the suppression of the expression of intracellular PPARγ and C / EBPα by 1,5-AF led to a decrease in the amount of accumulated fat by inhibiting the adipogenic enzyme.

Next, the influence of 1,5-AF on suppression of hypertrophy of mature adipocytes was examined. Even when 1,5-AF was added, the survival rate of mature adipocytes was the same as that of the control group, and thus 1,5-AF did not show cytotoxicity against mature adipocytes (FIG. 6). ). The amount of intracellular TG accumulation was reduced in a concentration-dependent manner by adding 1,5-AF in the range of 400 to 800 μg / ml (FIG. 7). In addition, many large lipid droplets of mature adipocytes stained by the Oil red O method are observed in the control, but when 1,5-AF is added, the intracellular lipid droplets are large. Became smaller and the number decreased. This effect was dependent on the added concentration of 1,5-AF (FIG. 8). Furthermore, the expression level of PPARγ, which is a transcription factor for fat synthesis, also significantly decreased depending on the concentration of 1,5-AF added (FIG. 9).
From the above results, it has been clarified that 1,5-AF suppresses the differentiation of preadipocytes and reduces the fat accumulation amount of mature adipocytes. Furthermore, these facts suggest that transcription factors such as PPARγ and C / EBPα are involved in being suppressed by 1,5-AF.

Claims (5)

An activity inhibitor of glycerol-3-phosphate dehydrogenase comprising 1,5-D-anhydrofructose. A production inhibitor of adipose synthesis transcription factor PPARγ and / or C / EBPβ comprising 1,5-D-anhydrofructose. The activity inhibitor of claim 1, wherein the glycerol-3-phosphate dehydrogenase is derived from a preadipocyte or a mature adipocyte. The production inhibitor according to claim 2, wherein the fat synthesis transcription factor PPARγ and / or C / EBPβ is derived from a preadipocyte or a mature adipocyte. The production inhibitor according to claim 2, which is accompanied by a reduction in the size of lipid droplets in mature fat cells.