JP5599020B2 - Mitochondrial fusion agent - Google Patents

Description

  The present invention relates to a mitochondrial fusion agent, and more particularly to a mitochondrial fusion agent containing a mitochondrial fusion peptide.

  As a result of an increase in the intake of calories and the amount of fat with the recent westernization of dietary habits, obesity and metabolic syndrome with accumulated fat have become social problems. Obesity and metabolic syndrome are likely to lead to serious diseases such as diabetes, hypertension, and arteriosclerosis. Drugs and foods with anti-obesity effects have been energetically developed.

  In obesity, fat accumulates in adipocytes, that is, an increase in the amount of triglyceride in the cells enlarges the fat cells, and the number of fat cells increases. In recent years, it has become clear that the number of fat cells increases even after adulthood. It is expected to suppress the progression of obesity and improve obesity by suppressing the differentiation from preadipocytes to mature adipocytes, reducing the number of mature adipocytes, and suppressing fat accumulation of mature adipocytes. The

  Conventionally, food ingredients such as β-conglycinin (Patent Document 1), isoflavone (Patent Document 2), and catechin (Patent Document 3) are known as substances having an anti-obesity effect. A peptide composed of Val-Val-Tyr-Pro is known to improve lipid metabolism so as to suppress an increase in blood triglyceride concentration (Patent Document 4).

  As a screening method for new substances with anti-obesity effect, differentiated adipocytes are cultured in the presence of the test substance, then the mitochondrial morphology in the cells is observed, and the mitochondria in the differentiated adipocytes are fragmented. In addition, there is a method of evaluating that the test substance is an anti-obesity agent when a tube-like network is formed without aggregation (Patent Document 5). In this method, it is possible to examine whether a test substance has an anti-obesity effect or a visceral fat accumulation suppression effect by giving a specific substance to a fat cell and observing the state of mitochondria.

JP 2005-80533 A WO02 / 004437 specification Japanese Patent Laid-Open No. 2003-95942 Japanese Patent No. 2805194 JP 2007-228855 A

  The present invention is to provide a mitochondrial fusion agent, an anti-obesity agent and a visceral fat accumulation inhibitor containing a novel mitochondrial fusion peptide as an active ingredient.

  As a result of investigating mitochondrial shape by giving various test substances to adipocytes, the present inventors have discovered a novel peptide having mitochondrial fusion activity, and using it as an active ingredient, a mitochondrial fusion agent, an anti-obesity agent and It came to produce for the visceral fat accumulation preventive agent. That is, the present invention provides a mitochondrial fusion agent containing Asp-Pro and containing a mitochondrial fusion peptide having 2 to 3 amino acids as an active ingredient.

  JP 2003-238589 describes Asp-Pro, Ala-Pro-Ser, Ala-Pro-Leu, and Ala-Pro-Lys as angiotensin converting enzyme inhibitory peptides. These peptides inhibit the conversion of angiotensin I to angiotensin II, which has a strong blood pressure raising effect, in the renin-angiotensin system. However, no anti-obesity action based on the mitochondrial fusion activity of Asp-Pro has been reported so far.

  The mitochondrial fusion peptide used in the present invention is preferably an Asp-Pro dipeptide.

  The present invention also provides an anti-obesity agent and visceral fat accumulation inhibitor containing the mitochondrial fusion peptide as an active ingredient.

  The present invention also provides a functional food or feed containing the mitochondrial fusion peptide and exhibiting mitochondrial fusion activity.

According to Kim et al. Of Korean University, H ⁺ -ATP synthase is present in the cell membrane of 3T3-L1 preadipocytes, and the enzyme increases as adipocytes differentiate (Exp. Mol. Med, Vol. 36, No. 5,476-485 (2004)). The mitochondria of undifferentiated 3T3-L1 preadipocytes form a long tubular network, but when preadipocytes differentiate into adipocytes, the structure of mitochondria becomes a fragmented or aggregated structure. That is, dynamic mitochondrial morphological changes (fragmentation) play an important role in inducing differentiation of adipocytes. As evidence, all the F1 inhibitors reported so far (compounds targeting F1 which is the catalytic site of mitochondrial H ⁺ -ATP synthase, such as resveratrol, peacetanol, kaempferol, etc.) are all lipid droplets in adipocytes. It was revealed that mitochondria were suppressed and mitochondria were induced in a tube shape. If mitochondrial fragmentation and aggregation can be suppressed and formed into a tube shape, induction of adipocyte differentiation is inhibited, leading to suppression of intracellular lipid droplets.

  The mitochondrial fusion peptide discovered by the present inventors promotes the formation of mitochondrial tubes and inhibits the accumulation of lipid droplets in adipocytes. Therefore, the mitochondrial fusion agent containing these peptides exhibits an anti-obesity action and a built-in fat accumulation inhibiting action. In addition to being useful as an anti-obesity agent and visceral fat accumulation preventing agent, the mitochondrial fusion agent of the present invention is also expected to be used as a muscle strength enhancer for sportsmen because it has an effect of increasing muscle mass. In addition, when the mitochondrial fusion peptide of the present invention is used for livestock animals, an effect of increasing meat mass and an effect of preventing excessive accumulation of lipids are expected.

A micrograph of mitochondria of a preadipocyte cultured in the presence of two mitochondrial fusion agents according to the present invention (drawing substitute photo) is shown along with a negative control cultured in the presence of PBS. In the figure, DP means Asp-Pro, DPC means Asp-Pro-Cys, and Control means negative control. All three photos are enlarged on the same scale. The micrograph (drawing substitute photograph) of the adipocyte cultured by adding two kinds of mitochondrial fusion agents according to the present invention is shown together with a negative control cultured in the presence of PBS. In the figure, DP means Asp-Pro, DPC means Asp-Pro-Cys, and Control means negative control. All three photos are enlarged on the same scale. The change of the visceral fat accumulation amount when two kinds of feeds to which the mitochondrial fusion agent of the present invention is added is administered to mice.

  Hereinafter, embodiments of the mitochondrial fusion agent, anti-obesity agent and visceral fat accumulation inhibitor (hereinafter referred to as mitochondrial fusion agent and the like) of the present invention will be described in detail.

  First, the mitochondrial fusion peptide which is an active ingredient such as the mitochondrial fusion agent of the present invention will be described. This peptide only needs to have mitochondrial fusion activity with 2 to 3 amino acids including Asp-Pro. A method for determining whether or not a peptide has mitochondrial fusion activity is described in detail in Japanese Patent Application Laid-Open No. 2007-228855, which is incorporated herein by reference. Specifically, after culturing differentiated adipocytes in the presence of substances that are candidates for mitochondrial fusion peptides, the mitochondrial morphology in the cells is observed, and mitochondria in the differentiated adipocytes are not fragmented and aggregated. Next, it is examined whether a tube-like network is formed. At that time, the form of mitochondria when differentiated adipocytes are cultured in the absence of the test substance is used as a negative control. The stronger the activity of making mitochondria into a tube, the higher the anti-obesity activity.

  The mitochondrial fusion peptide may be obtained from a protein degradation product or may be obtained by chemical synthesis. Alternatively, a peptide can also be prepared by preparing cDNA consisting of a base sequence corresponding to the amino acid sequence of the peptide and then incorporating it into an appropriate vector and expressing the gene in an appropriate host.

  When obtaining a mitochondrial fusion peptide from a protein degradation product, animal-derived proteins such as casein, whey, fish protein, and egg white, and plant-derived proteins such as soybean, soybean germ, wheat and corn can be widely used as raw materials. .

  As a method for degrading proteins, there are a method using an enzyme, a method using an acid or an alkali, and the like. When using an enzyme, any of acidic protease, neutral protease, or alkaline protease may be used. If necessary, two or more kinds of proteases may be used. Arbitrary acids and alkalis can also be used.

  The proteolysate obtained above may be used as it is, or may be used after purification. Purification can be performed by appropriately combining known purification steps such as concentration, precipitation, desalting, separation by adjusting salt concentration and pH, ion exchange resin method, ultrafiltration method, reverse phase chromatography method and the like.

  Mitochondrial fusion peptides can also be chemically synthesized by known peptide synthesis methods. Examples thereof include an azide method, an acid chloride method, an acid anhydride method, a mixed acid anhydride method, a DCC method, an active ester method, a carboimidazole method, and a redox method. These peptide synthesis methods can be performed by either solid phase synthesis method or liquid phase synthesis method.

  In the method for synthesizing mitochondrial fusion peptides, amino acids having a side chain functional group, such as tyrosine or threonine, preferably protect the side chain functional group. As the protecting group, a known protecting group such as a benzyloxycarbonyl group, a t-butoxycarbonyl group, a benzyl group or the like can be used, and this protecting group can be deprotected by a known method.

  The mitochondrial fusion agent or the like of the present invention may be one using one or more of the mitochondrial fusion peptides alone. Moreover, the composition which mix | blended the adjuvant and food additive which can be used pharmacologically according to final products, such as a pharmaceutical, a functional food, health supplement food, and food-drinks, may be sufficient. The form of the mitochondrial fusion agent of the present invention may be any of solid, liquid, sol, gel, plastic composition and the like.

  Specific examples of the pharmacologically usable adjuvant include glucose (glucose), maltose, fructose (fructose), galactose, trehalose, oligosaccharide, sorbit, lactose, sucrose, sucrose, purified sucrose, erythritol, xylitol, Sugars such as sorbitol, mannitol, palatinose, reduced palatinose, powdered reduced maltose, water candy, carmellose, dextrin; corn starch, pregelatinized starch, partially pregelatinized starch, potato starch, corn starch, hydroxypropyl starch, amino acids, kaolin, anhydrous silica Acid, silicic acid, aluminum silicate, sodium bicarbonate, calcium phosphate, calcium dihydrogen phosphate, calcium carbonate, magnesium oxide, aluminum hydroxide, fatty acid or salt thereof, fatty acid Noglycerides and diglycerides, alcohols, viscous paraffins, propylene glycol, ethylene glycol, polyethylene glycol, glycerin and other carriers or excipients; crystalline cellulose, crystalline cellulose / carmellose sodium, methylcellulose, hydroxypropylcellulose, low substituted hydroxypropylcellulose, Hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, carmellose sodium, ethylcellulose, carboxymethylethylcellulose, hydroxyethylcellulose, dextrin, pullulan, polyvinylpyrrolidone, aminoalkyl methacrylate copolymer E, aminoalkyl methacrylate copolymer Binders such as Mer RS, methacrylic acid copolymer L, methacrylic acid copolymer, polyvinyl acetal diethylaminoacetate, polyvinyl alcohol, gum arabic, gum arabic powder, agar, gelatin, white shellac, tragacanth, macrogol; synthetic aluminum silicate, dry water Aluminum oxide gel, magnesium aluminate metasilicate, calcium hydrogen phosphate, anhydrous calcium hydrogen phosphate, waxes, hydrogenated vegetable oil, polyethylene glycol, light anhydrous silicic acid, synthetic aluminum silicate, stearic acid, macrogol, talc, stearin Lubricants such as magnesium oxide, calcium stearate, hydrous silicon dioxide, sucrose fatty acid ester; lubricant; crystalline cellulose, methylcellulose, low-substituted hydroxypropylcellulose, Disintegrants such as carmellose, carmellose calcium, carmellose sodium, croscarmellose sodium, wheat starch, rice starch, corn starch, potato starch, partially pregelatinized starch, hydroxypropyl starch, sodium carboxymethyl starch, tragacanth; soy lecithin, Sucrose fatty acid ester, polyoxyl stearate, polyoxyethylene hydrogenated castor oil, polyoxyethylene polyoxypropylene glycol, sorbitan sesquioleate, sorbitan trioleate, sorbitan monostearate, sorbitan monopalmitate, sorbitan monolaurate, polysorbate, Surfactants such as glyceryl monostearate, sodium lauryl sulfate, lauromacrogol; phosphorus Solubilizing agents such as sodium; lactic acid, gluconic acid, succinic acid, fumaric acid, citric acid, L malic acid, DL malic acid, glacial acetic acid, acetic acid, glucono delta lactone, L tartaric acid, DL tartaric acid, hydrochloric acid, sodium citrate PH adjusters such as sodium hydroxide, potassium hydroxide, sodium hydrogen carbonate, sodium carbonate; absorption accelerators; brighteners such as natural resins; emulsifiers; stabilizers; antioxidants; preservatives; A soothing agent and the like.

  Examples of the food additive include cereal-based powders such as starch, fats and oils, emulsifiers, thickeners, and fragrances. Examples of the starch include corn starch, waxy corn starch, high amylose corn starch, potato starch, wheat starch, tapioca starch, mung bean starch, sago starch, rice starch, pea starch, and esterification treatment, etherification treatment, Mention may be made of processed starch which has been subjected to physical or chemical treatment such as cross-linking treatment, acid treatment, oxidation treatment, wet heat treatment and pregelatinization alone or in combination.

  Examples of the fats and oils are soybean oil, soybean germ oil, rapeseed oil, high oleic rapeseed oil, corn oil, sesame oil, sesame salad oil, perilla oil, linseed oil, peanut oil, safflower oil, safflower oil, high oleic safflower oil, sunflower oil, High oleic sunflower oil, high linoleic sunflower oil, mid oleic sunflower oil, cottonseed oil, grape seed oil, macadamia nut oil, hazelnut oil, walnut oil, pumpkin seed oil, coconut oil, tea seed oil, sesame oil, olive oil, rice bran oil , Wheat germ oil, palm oil, palm olein, palm kernel oil, coconut oil, cacao butter, algae oil, and hydrogenated oil, transesterified oil, fractionated oil, etc. Can be mentioned.

  Examples of the emulsifier may be any emulsifier usually used for foods, such as glycerin fatty acid ester, glycerin organic acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester, polyglycerin fatty acid ester, polyglycerin condensed ricinolate, Use sugar fatty acid ester, calcium stearoyl lactate, alkylglycosidic acid, erythritol fatty acid esters, polyoxyethylene sorbitan fatty acid esters, lecithin, enzymatically decomposed lecithin, enzyme-treated lecithin, etc., alone or in combination of two or more. Can do.

  As an example of the fragrance, any of natural and synthetic fragrances usually used in foods can be used. Thickeners include polysaccharides that increase viscosity when made into aqueous solutions, i.e. gum arabic, arabinogalactan, guar gum, xanthan gum, psyllium seed gum, gellan gum, tara gum, locust bean gum, tamarind seed gum, water soluble in soybeans Examples include polysaccharides (hemicellulose), sodium alginate, pullulan, pectin, karaya gum, gati gum, tragacanth gum, curdlan, glucomannan, chitin, chitosan, microfibrous cellulose, and microcrystalline cellulose.

  In addition, collagen peptides, milk protein peptides, casein peptides, oligopeptides, milk protein concentrates, pea protein, protein-derived substances such as gelatin, soy fiber, fiber such as pea fiber, highly branched cyclic dextrin Dextrins such as can also be added.

  The amount of mitochondrial fusion peptide in the mitochondrial fusion agent and the like of the present invention is appropriately adjusted depending on the use (pharmaceutical or functional food) of the composition, the intake, the intake method, and the like. Content may be 0.0001-100 weight% normally, Preferably it is 0.001-90 weight%, More preferably, it is 0.01-70 weight%, More preferably, it is the range of 1-50 weight%. . If the peptide content is 0.0001% by weight or less, it may not be possible to take in an amount necessary to obtain an anti-obesity effect.

  The dosage and usage as a pharmaceutical of the mitochondrial fusion agent of the present invention can be determined in consideration of various factors that affect the patient's symptoms, body weight, administration interval, administration method, and other clinical effects. When used for the purpose of obesity prevention, the daily intake amount for an adult male may usually be 0.01 mg to 10000 mg, preferably 0.1 mg to 500 mg. When used for therapeutic purposes, 1 mg to 10000 mg can be used.

  The intake method when using the mitochondrial fusion agent of the present invention as a medicine is not particularly limited. For example, oral ingestion, transdermal administration, infusion, injection (intramuscular, intraperitoneal, subcutaneous, or intravenous). Preferably, the tablet or capsule is taken orally in that the administration burden is small.

  When the mitochondrial fusion agent of the present invention is used in functional foods, health foods, supplements or general foods, the dosage per day for an adult male is preferably 0.01 mg to 10000 mg, preferably 0.1 to 500 mg is more preferable.

  The mitochondrial fusion agent of the present invention may be used as a medicine or functional food to be ingested by animals other than humans such as domestic animals and pets. Administration methods include parenteral intake such as injection, and oral intake in the form of functional foods and mixed feeds.

Hereinafter, the present invention will be described in more detail with reference to examples. The present invention is not limited to the following examples.
[Examples 1 and 2]
Asp-Pro dipeptide (Example 1) and Asp-Pro-Cys (Example 2), which is a tripeptide having Asp-Pro as a basic skeleton, were chemically synthesized. The mitochondrial fusion activity of these peptides was analyzed by the following procedure based on the method described in Japanese Patent Application Laid-Open No. 2007-228855.

1.3 Culture of T3-L1 cells Confluent undifferentiated 3T3-L1 preadipocytes in a basal medium consisting of Dulbecco's Modified Eagle Medium containing 10% Fetal bovine serum, glucose, kanamycin and biotin Until cultured. This was used for the observation of mitochondrial morphology as 3T3-L1 preadipocytes (Day 0).

  Subsequently, undifferentiated 3T3-L1 preadipocytes are cultured for about 2 days in a differentiation induction medium in which 0.25 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 2 μM insulin are added to the basal medium, and further 2 μM. The cells were cultured in a differentiation induction medium containing insulin for about 14 days to differentiate into adipocytes. This was used for observation of visceral fat accumulation as 3T3-L1 adipocytes (Day 16).

2. Measurement of mitochondrial fusion activity The supernatant of 3T3-L1 preadipocytes (Day 0) was removed and transferred to the culture medium. The test substances shown in Table 1 were diluted in PBS, added to the culture medium, and cultured for 48 hours. Mitochondria were stained with MitoTracker Green FM (Molecular Probes) at 37 ° C. for 30 minutes for mitochondrial morphological changes. Using a 100 × objective lens of a fluorescence microscope (OLYMPUS IX70, manufactured by Olympus), mitochondrial morphology was observed by oil immersion. FIG. 1 shows a photomicrograph of mitochondria of preadipocytes cultured with addition of Asp-Pro or Asp-Pro-Cys, together with a negative control cultured in the presence of PBS.

Photographs of 10 to 20 bright-field images and 10 to 20 fluorescent images of cells stained with mitochondria for fluorescence were taken, and mitochondrial morphology of about 50 cells was recorded. Using MetaMorph, which is a photography and image analysis software, a scale bar (10 μm) was inserted into a stored photograph, and the length of mitochondria was measured visually. The mitochondrial length for each individual cell was classified according to the following criteria and scored. A score was calculated for each photo and cell, and the total was taken as the mitochondrial fusion level of the sample.
(1) Granule type (1 μm or less: score-1)
(2) Short tube type (1-2 μm: score + 1)
(3) Medium tube type (2-5 μm: score + 2)
(4) Long tube type (5 μm or more: score + 3)
(5) Highly tube type (5 μm or more and highly tubed: score + 5)

  Table 1 shows the results of setting the negative control mitochondrial fusion level to 100. From Table 1, it was proved that the tripeptide having Asp-Pro dipeptide and Asp-Pro as a basic skeleton has mitochondrial fusion activity.

3. Measurement of activity of inhibiting fat accumulation in cells 3T3-L1 adipocytes (Day 16) supernatant was removed, transferred to a differentiation maintenance medium, and a test substance (Table 1) diluted in PBS was further added and cultured for 48 hours. did. Here, the concentration of the test substance was 0.1 μg / ml. PBS was used as a negative control. Intracellular lipid droplets were stained with Oil-Red O, the amount of lipid droplets was calculated, and the influence on lipid droplet accumulation was analyzed. First, adipocytes were observed with a 40 × objective lens of a microscope (OLYMPUS IX70 manufactured by Olympus). FIG. 2 shows micrographs of adipocytes cultured with Asp-Pro or Asp-Pro-Cys added together with negative controls.

10 to 20 bright field images of the cells were taken, and the diameter of the lipid droplets was measured using MetaMorph. At that time, the diameter of the lipid droplet for each cell was classified according to the following criteria and scored. A score was calculated for each photo and cell, and the total was taken as the intracellular fat level of the sample.
(1) Small type (less than 2 μm: score 0)
(2) Medium type (2-5 μm: score +1)
(3) Large type (5 μm or more: score +2)

  Table 1 shows the results of setting the intracellular lipid droplet level of the negative control (PBS) to 100. From the results of Table 1, it was proved that the mitochondrial fusion peptide composed of Asp-Pro and the mitochondrial fusion peptide composed of a tripeptide having Asp-Pro as a basic skeleton have an inhibitory effect on visceral fat accumulation.

[Example 3] (Administration test to mice)
Using a 4-week-old C57BL / JJcl strain male mouse (manufactured by CLEA Japan, Inc.), a mitochondrial fusion agent administration test comprising Asp-Pro was conducted. First, the animals were acclimated for 1 week, and then divided into 5 groups per group as follows.
(1) SD group: normal food (trade name: CE-2, manufactured by CLEA Japan, Inc.) and water (2) SD + DP group: normal food and water mixed with Asp-Pro (3) HF Group: High fat diet (trade name: High Fat Diet 32 (HFD32), manufactured by CLEA Japan) and water (4) HF + DP group: High fat diet and water mixed with Asp-Pro

  As for the dose of Asp-Pro, the daily water intake of the mouse was measured in advance, and the Asp-Pro concentration was adjusted so that 3 mg was administered per kg body weight of the mouse. The administration period was 1 week.

  Table 2 shows the data of body weight before and after the administration test, water consumption and food intake during the administration period. The group given Asp-Pro mixed water tended to increase food intake and water intake compared to the group given normal water. In a comparison between the HF group and the HF + DP group that ingested a high fat diet, there was a tendency for the body weight change to decrease when drinking Asp-Pro-mixed water.

  FIG. 3 shows the results of measuring the amount of visceral fat by dissecting the mice after the administration. In both the normal diet and the high fat diet, the amount of visceral fat was reduced by administration of Asp-Pro mixed water. From Table 2 and FIG. 3, it was proved that the mitochondrial fusion agent comprising Asp-Pro has an anti-obesity effect and a visceral fat accumulation inhibitory effect.

Claims (5)

A mitochondrial fusion agent containing, as an active ingredient, a mitochondrial fusion peptide comprising at least one of Asp-Pro and Asp-Pro-Cys . An anti-obesity agent comprising a mitochondrial fusion peptide comprising at least one of Asp-Pro and Asp-Pro-Cys as an active ingredient. A visceral fat accumulation inhibitor containing, as an active ingredient, a mitochondrial fusion peptide comprising at least one of Asp-Pro and Asp-Pro-Cys .   A functional food containing a mitochondrial fusion peptide composed of Asp-Pro-Cys and exhibiting mitochondrial fusion activity.   A feed containing a mitochondrial fusion peptide comprising Asp-Pro-Cys and exhibiting mitochondrial fusion activity.