JP2013035763A - Fat accumulation suppressor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a material capable of suppressing fat cell differentiation and maturation or fat accumulation and effective for prevention and amelioration of symptoms due to adiposity.SOLUTION: A fat cell differentiation and maturation and/or fat accumulation suppressor includes, as an effective ingredient, a C-terminus amidated polypeptide comprising an amino acid sequence wherein 0-3 amino acids are connected to the N-terminus side of an amino acid sequence represented by a sequence number 1.

Description

  The present invention relates to an adipocyte differentiation / maturation inhibitor, fat accumulation inhibitor, and obesity prevention and / or amelioration agent.

  In recent years, the obese population has been increasing due to westernization of eating habits, lack of exercise and stress. Obesity has been reported to cause various lifestyle-related diseases such as insulin resistance and arteriosclerosis, and is a major social problem in terms of maintaining and promoting the health of the people.

  Obesity refers to a state in which body fat has accumulated excessively, and is caused by the amount of energy consumed exceeding the amount of energy consumed. Therefore, reducing the amount of energy intake or increasing the amount of energy consumed is thought to lead to the prevention and improvement of obesity. Examples of methods for preventing and improving obesity and various lifestyle-related diseases associated with obesity include exercise, calorie restriction, and improvement of lifestyle habits. These are generally considered effective for the prevention and amelioration of obesity, but in many cases sufficient effects cannot be obtained in many cases.

  So far, extensive research has been conducted with the aim of developing obesity-improving agents and lipid metabolism promoters. For example, for the purpose of promoting energy metabolism, PPAR (Peroxisome Proliferator Activated Receptor: Peroxisome proliferator activated receptor) activator (Non-patent Documents 1 to 3) or inhibitor (Non-patent Document 4), AMPK (AMP-activated protein kinase) activator (Non-patent Documents 5 and 6) and the like. In addition, ACC-1 (acetyl-CoA carboxylase) inhibitor (Non-patent Document 7), FAS (Fatty Acid Synthase) inhibitor (Patent Document 1), and the like can be cited as the purpose of suppressing fat synthesis. However, these effects are not always sufficient.

  Obesity is also closely linked to adipocyte hypertrophy and an increase in cell number, and therefore suppresses the differentiation of preadipocytes into mature adipocytes, or suppresses fat accumulation in adipocytes. Is considered effective in the treatment and prevention of obesity. Insulin stimulation, dexamethasone and isobutylmethylxanthine are known as induction stimuli for differentiating fat cells into mature fat cells. With these differentiation-inducing stimuli, the expression of transcription factors, PPARs and C / EBPs, which are master regulators of adipocyte differentiation, increased, followed by ACC-1, FAS and DGAT (diacylglycerol acyltransferase) and lipid synthesis involved in lipid synthesis. Furthermore, the expression of various lipid metabolism-related genes specifically expressed in mature adipocytes such as GLUT4 (glucose transporter 4) and LPL (lipoprotein lipase) involved in sugar / lipid uptake increases. Further, Fabp4 (fatty acid binding protein 4), which is a fatty acid transport carrier, is also known as a differentiation maturation marker for adipocytes that are specifically expressed in differentiated adipocytes.

  As with other cell types, adipocytes are surrounded by an extracellular matrix (ECM) around the cells. It has been reported that ECM not only provides a scaffold for cell adhesion, but also contributes to many physiological activities such as cell proliferation, differentiation, and migration via receptors present on the cell membrane surface. The molecular composition of ECM varies from cell to cell, and cells are thought to express and maintain cell functions in an appropriate state by forming an environment called ECM having a specific molecular composition.

Laminin is a protein known as a major component of the basement membrane, which is an ECM that exists between the epithelial cell layer and the stromal cell layer. Laminin forms a heterotrimer composed of α, β, and γ chains, and plays a central role in cell adhesion to the basement membrane. Laminin is also known to have many biological activities such as cell proliferation, migration and neurite outgrowth.
The activity of laminin is thought to be exerted through the laminin signal system through the binding of laminin to a cell surface receptor and intracellular signaling coupled thereto. Integrins α6β1, α3β1, α6β4, α7β1, dystroglycan, syndecan and the like are known as main receptors for receiving laminin (Non-patent Document 8).

  Laminin is present in various tissues and is also expressed in adipose tissue. It has been reported that laminin-8 (α4β1γ1) is particularly expressed in adipocytes, and that gene expression increases with differentiation and maturation (Non-patent Document 9). However, the expression level change and physiological significance of laminin in adipose tissue have not yet been fully clarified. In addition, the effect of laminin on adipocyte properties such as differentiation maturation and fat accumulation has not been elucidated.

JP 2007-63156 A

Curr. Opin. Lipidol., 1999, 10: 151-159 BMC Pharmacol., 2004, 4 (23) Cell 113, 2003: 159-170 Folia. Pharmacol. Jpn., 2003, 122: 294-300 Molecular Medicine., 2002, 39 (4): 398-407 Diabetes., 2001, 50 (5): 1076-1082 Nat.Med., 2000, 6 (10): 1115-1120 Matrix Biology, 2006, 25 (3): 189-197 Matrix Biology, 1997, 16 (4): 223-230

  TECHNICAL FIELD The present invention relates to an adipocyte differentiation / maturation inhibitor and / or fat accumulation inhibitor, and an obesity prevention and / or amelioration agent comprising a laminin partial peptide as an active ingredient.

  The present inventors investigated the effect of the extracellular environment containing the extracellular matrix on adipocyte characteristics. As a result, laminin has the effect of enhancing adipocyte differentiation and maturation and fat accumulation, and this effect is blocked by a peptide having an action of inhibiting laminin-receptor binding, thereby adipocyte differentiation and maturation and fat accumulation. It was found that accumulation can be suppressed.

That is, the present invention provides the following.
(1) An adipocyte differentiation / maturation inhibitor comprising, as an active ingredient, a C-terminal amidated product of a polypeptide comprising an amino acid sequence having 0 to 3 amino acids bonded to the N-terminal side of the amino acid sequence represented by SEQ ID NO: 1 And / or a fat accumulation inhibitor.
(2) The adipocyte differentiation / maturation inhibitor and / or fat accumulation inhibitor according to (1), wherein the polypeptide comprises the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.
(3) The adipocyte differentiation / maturation inhibitor and / or fat accumulation inhibitor according to any one of (1) and (2), wherein the C-terminal amidated form of the polypeptide is bound to polyethylene glycol.
(4) An obesity preventive and / or ameliorating agent comprising as an active ingredient a C-terminal amidated form of a polypeptide comprising an amino acid sequence in which 0 to 3 amino acids are bonded to the N-terminal side of the amino acid sequence represented by SEQ ID NO: 1. .
(5) The obesity preventing and / or improving agent according to (4), wherein the polypeptide consists of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.
(6) The obesity preventive and / or ameliorating agent according to (4) or (5), wherein the C-terminal amidated form of the polypeptide is bound to polyethylene glycol.
(7) A pharmaceutical or quasi-pharmaceutical product comprising as an active ingredient a C-terminal amidated form of a polypeptide comprising an amino acid sequence having 0 to 3 amino acids bonded to the N-terminal side of the amino acid sequence represented by SEQ ID NO: 1.
(8) An external preparation comprising as an active ingredient a C-terminal amidated product of a polypeptide comprising an amino acid sequence in which 0 to 3 amino acids are bonded to the N-terminal side of the amino acid sequence represented by SEQ ID NO: 1.

  The present invention can suppress differentiation and maturation of fat cells or fat accumulation, thereby reducing body fat systemically or locally, and further obesity, diabetes, hyperlipidemia, hypertension caused by body fat accumulation, To provide materials that are effective in preventing and improving symptoms such as cardiovascular diseases.

Effect of laminin on differentiation and maturation of adipocytes: Influence of laminin on the expression level of genes specifically expressed in differentiated adipocytes. LM; laminin, *; P <0.05, **; P <0.01, ***; P <0.001 (Student's t-test), error bar = standard error, n = 3. Laminin promotes fat accumulation. A: Oil-red O stained adipocytes, B: Cell staining (relative absorbance of isopropanol extract of Oil-red O stained cells). LM; laminin, *; P <0.05 (Student's t-test), error bar = standard error, n = 3. Laminin partial peptide used in Examples. Inhibition of Fabp4 expression in adipocytes by laminin partial peptide. **; P <0.01 (Student's t-test).

  In the present specification, “non-therapeutic” is a concept that does not include a medical act, that is, a treatment act on the human body by therapy.

  As used herein, “prevention” refers to preventing or delaying the onset of a disease or symptom in an individual, or reducing the risk of developing an individual's disease or symptom.

  In the present specification, “improvement” refers to improvement of a disease or symptom, prevention or delay of worsening of the disease or symptom, or reversal, prevention or delay of progression of the disease or symptom.

  In this specification, a laminin signal refers to a series of processes in which a signal is transmitted into a cell via laminin receptors present on the surface of a cell membrane from laminin, which is a kind of extracellular matrix (ECM). That is, the laminin signal can be, for example, the expression level of laminin or laminin receptor by a cell, the binding between laminin receptor and laminin on the cell surface, intracellular signal transduction caused by laminin, and the like.

As shown in a reference example described later, laminin has an action of promoting differentiation / maturation of adipocytes and an action of promoting fat accumulation in fat cells. That is, a substance capable of suppressing cellular signal transduction (laminin signal) and subsequent cellular activity caused by laminin is a substance that has the effect of inhibiting differentiation and maturation of adipocytes and inhibiting fat accumulation in adipocytes. Useful as.
As a method for suppressing laminin signal, a method of competitively inhibiting the binding of laminin and integrin with a peptide or a neutralizing antibody against laminin or its receptor, or negatively controlling integrin signal, or laminin or Although the method of reducing the expression level of laminin in a fat cell or its receptor using siRNA with respect to the receptor, etc. is mentioned, It is not limited to this.

  Further, as shown in the Examples below, the C-terminal amidated form of a polypeptide having a specific sequence derived from a laminin partial peptide is caused by laminin by inhibiting the binding of laminin to cellular laminin receptors. It has the effect of suppressing the progression of the differentiation and maturation process of adipocytes. Therefore, the C-terminal amidated form of the laminin partial peptide and its equivalent laminin-receptor binding inhibitory peptide and its modified form inhibit the accumulation of fat in fat cells in order to suppress the differentiation and maturation of fat cells. Therefore, it can be used for body fat accumulation suppression or for prevention and / or improvement of symptoms such as obesity, diabetes, hyperlipidemia, hypertension, cardiovascular disease and the like caused by body fat accumulation. The use can be in humans or non-human animals, or specimens derived therefrom, and can be therapeutic or non-therapeutic.

Accordingly, the present invention provides an adipocyte differentiation comprising as an active ingredient a C-terminal amidated product of a polypeptide comprising an amino acid sequence having 0 to 3 amino acids bonded to the N-terminal side of the amino acid sequence represented by SEQ ID NO: 1. A maturation inhibitor and / or a fat accumulation inhibitor is provided.
The present invention also provides an anti-obesity and / or an active ingredient comprising a C-terminal amidated product of a polypeptide comprising an amino acid sequence in which 0 to 3 amino acids are bonded to the N-terminal side of the amino acid sequence represented by SEQ ID NO: 1. Provide an improver.
In one embodiment, the agent may consist essentially of only the C-terminal amidated form of the polypeptide.

The C-terminal amidated form of the polypeptide used as the active ingredient of the present invention has 0 to 3 amino acid residues in the N-terminal direction of the amino acid sequence YIGSR (Tyr-Ile-Gly-Ser-Arg: SEQ ID NO: 1). in the polypeptide consisting of the peptide binding amino acid sequence, a carboxyl group -COOH of C-terminal amino acid residues, which is amidated has become -CONH _2, and the binding of the laminin receptor laminin and cell Any material having an inhibitory effect may be used. That is, the C-terminal amidated form of the polypeptide used as the active ingredient of the present invention consists of an amino acid sequence represented by (X) n-YIGSR (X is an arbitrary amino acid residue, n is an integer of 0 to 3). It is a C-terminal amidated form of a polypeptide. Examples of the polypeptide include a polypeptide consisting of SEQ ID NO: 1 and a polypeptide consisting of SEQ ID NO: 2 (Asp-Pro-Gly-Tyr-Ile-Gly-Ser-Arg).

  The polypeptide represented by SEQ ID NO: 1 corresponds to a laminin fragment consisting of amino acids 998-1002 of mouse laminin β1 chain, or a laminin fragment consisting of amino acids 950-954 of human laminin β1 chain. The amino acid sequences of mouse and human laminin β1 chain are registered in the database (NCBI) as (NM_008482) and (NM_002291), respectively. Therefore, the polypeptide used in the present invention can be identified by selecting an arbitrary region containing the amino acid sequence represented by SEQ ID NO: 1 based on the information in the database.

  As long as the partial peptide of laminin selected as described above contains the polypeptide represented by SEQ ID NO: 1 and has an action of inhibiting the binding between laminin and cellular laminin receptor, the amino acid is further mutated. May be. For example, 1 to 3, preferably 1 to 2, more preferably 1 amino acid may be mutated. Here, mutation means amino acid deletion, substitution, or addition.

  The C-terminal amidated form of the polypeptide used in the present invention can be obtained from any polypeptide according to a known method such as a biochemical method or an organic synthetic method using a C-terminal α-amidating enzyme or the like. It can be prepared by amidating the C-terminus. Alternatively, C-terminal amidated polypeptides can be purchased from Biomatik Corporation, Greiner-Bio One, and the like.

  The C-terminal amidated product of the polypeptide may be modified. The C-terminal amidated form of the modified polypeptide can be obtained by any means commonly used in the art as long as the action of inhibiting the binding between laminin and the receptor is not essentially lost. It may be modified. Preferable examples include a modified product in which polyethylene glycol is bound to the C-terminal amidated product of the polypeptide of SEQ ID NO: 1.

The C-terminal amidated form of the above polypeptide is also used for inhibiting differentiation and maturation of fat cells, for suppressing fat accumulation in fat cells, for suppressing fat accumulation, or for obesity, diabetes, It can be used for the manufacture of a composition for preventing and / or improving symptoms such as lipemia, hypertension, cardiovascular disease, etc., pharmaceutical, quasi-drug, external preparation, food and drink, feed and the like.
These compositions, medicines, quasi drugs, external preparations, foods and drinks, feeds and the like can be produced or used for human or non-human animals. The C-terminal amidated form of the above polypeptide is blended in the composition, medicine, quasi-drug, external preparation, food / drink, feed, etc., and suppresses fat accumulation in fat cells to suppress adipocyte differentiation and maturation. Therefore, it may be an active ingredient for suppressing body fat accumulation or for preventing and / or improving symptoms such as obesity, diabetes, hyperlipidemia, hypertension, cardiovascular disease and the like caused by body fat accumulation. The composition, medicine, quasi drug, external preparation, food and drink, feed and the like are also within the scope of the present invention.

  The medicine, quasi-drug, or external preparation contains a C-terminal amidated form of the polypeptide as an active ingredient. The pharmaceutical or quasi drug can be administered in any dosage form. Administration may be oral or parenteral, but oral administration is preferred. Dosage forms for oral administration include, for example, solid dosage forms such as tablets, coated tablets, granules, powders, capsules, and liquid dosage forms such as elixirol, syrups and suspensions, Examples of the dosage form for parenteral administration include injection, infusion, transdermal, transmucosal, nasal, enteral, inhalation, suppository, bolus, patch and the like. Examples of the dosage form of the external preparation include ointment, lotion, gel, cream, patch, spray and the like.

  The pharmaceutical, quasi-drug or external preparation may contain the C-terminal amidated form of the polypeptide alone or in combination with a pharmaceutically acceptable carrier. Examples of such carriers include excipients, coating agents, binders, extenders, disintegrating agents, lubricants, diluents, surfactants, osmotic pressure adjusting agents, pH adjusting agents, dispersing agents, emulsifiers, antiseptics. Agents, stabilizers, antioxidants, colorants, ultraviolet absorbers, humectants, thickeners, activity enhancers, anti-inflammatory agents, bactericides, fragrances, flavoring agents, flavoring agents and the like. In addition, the drug, quasi-drug, and external preparation contain other active ingredients and pharmacological ingredients as long as the inhibitory action on laminin-receptor binding of the C-terminal amidated form of the polypeptide is not lost. Also good.

  The said food-drinks or feed contains the C-terminal amidation body of the said polypeptide as an active ingredient. The food / drink or feed of the present invention is a fat cell differentiation / maturation suppression, fat cell fat accumulation suppression, body fat accumulation suppression, or obesity due to body fat accumulation, diabetes, hyperlipidemia, hypertension, cardiovascular disease Intended for functions such as prevention and / or improvement of symptoms such as beauty foods, nutritional function foods, sick foods, foods for specified health use, etc. Functional food or drink or feed.

The food or drink may contain a C-terminal amidated product of the polypeptide alone or as long as the inhibitory action on laminin-receptor binding of the C-terminal amidated product of the polypeptide is not lost. , May contain other food ingredients and additives such as solvents, softeners, oils, emulsifiers, preservatives, fragrances, stabilizers, colorants, antioxidants, humectants, thickeners, etc. .
Examples of the beverage include all beverages such as fruit juice beverage, carbonated beverage, tea-based beverage, coffee beverage, near water, sports beverage, milk beverage, alcoholic beverage, and soft drink. Moreover, a drink can be used as the container-packed drink with which the container was filled.
The form of the food may be any form such as solid, semi-solid, liquid, etc., and may be tablet form, pill form, tablet, capsule form, liquid form, syrup form, powder form, granule form, etc. Also good. For example, as food, in addition to various foods such as breads, cakes, noodles, pasta, confectionery, jelly, various snacks, frozen foods, ice creams, dairy products, beverages, soups, edible oils, Examples include the same forms (tablets, capsules, syrups, etc.) as the above-mentioned oral administration preparations.

  The feed may contain the C-terminal amidated product of the polypeptide alone or as long as the inhibitory action on laminin-receptor binding of the C-terminal amidated product of the polypeptide is not lost. Commonly used feed ingredients such as meat such as cattle, pigs, sheep, protein, grains, bran, potatoes, sugars, vegetables, vitamins, minerals, etc. A mold, a pH adjuster, a seasoning, a preservative, a nutritional reinforcing agent and the like may be contained as necessary. As feed, for example, livestock feed used for cattle, pigs, chickens, sheep, horses, etc., feed for small animals used for rabbits, rats, mice, etc., feed for seafood used for tuna, eel, Thailand, hamachi, shrimp, Examples include pet food used for dogs, cats, small birds, squirrels, and the like.

The pharmaceutical, quasi-drug, external preparation, food and drink, or feed is obtained from the C-terminal amidated form of the polypeptide or, if necessary, the carrier, additive, or other active ingredient, pharmacological ingredient, foodstuff or The raw materials can be combined and manufactured by a conventional method.
The content of the polypeptide in the medicine, quasi-drug, external preparation, food / beverage product or feed is usually 0.00001 to 10% by mass, and preferably 0.0001 to 5% by mass.

  The present invention also provides a method for suppressing differentiation maturation and / or fat accumulation of adipocytes. The method includes a step of adding or administering a C-terminal amidated form of the polypeptide to adipocytes. Examples of administration subjects include animal-derived adipose tissue, adipocytes, or cultures thereof, and animals that require the differentiation and maturation of adipocytes and / or suppression of fat accumulation. The animal is preferably a human or non-human mammal, more preferably a human.

  The present invention also provides a method for inhibiting body fat accumulation or preventing and / or improving symptoms such as obesity, diabetes, hyperlipidemia, hypertension and cardiovascular disease caused by body fat accumulation. The method includes a step of administering a C-terminal amidated form of the polypeptide to an animal in need of suppressing body fat accumulation or preventing and / or improving the disease. The animal is preferably a human or non-human mammal, more preferably a human. In a preferred embodiment, the method is a method for preventing and / or improving obesity.

  In the method of the present invention described above, when the subject is a cultured cell, the concentration at which the C-terminal amidated form of the polypeptide is added or administered is 0.0005 to 20% (w / v) as the final concentration in the culture. ), Preferably 0.005 to 10% (w / v), when administered to an adult, 0.0001 to 10% per day for external use, preferably 0.0005 to 5%, orally administered In the case, it may be 0.0001-20 mg / kg, preferably 0.0005-10 mg / kg. In one aspect, the method of the present invention described above may be a non-therapeutic method based on the concept of slimming for cosmetic purposes.

  EXAMPLES Hereinafter, an Example is shown and this invention is demonstrated more concretely.

Reference Example 1 Adipocyte differentiation and maturation promoting effect of laminin A mouse preadipocyte-derived cell line 3T3-L1 cell (ATCC) was used for the experiment. As a normal medium, high glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum was used, and cultured under conditions of 37 ° C. and 5% CO ₂ .
3T3-L1 cells were seeded in a 6-well dish (BD) coated with laminin, normally cultured until 100% confluent, and then the medium was replaced with a differentiation-inducing medium. The differentiation induction medium used was a medium supplemented with 5 μg / ml insulin, 1 μM dexamethasone, and 0.115 mg / ml isobutylmethylxanthine. The day when the medium was changed to the differentiation-inducing medium was defined as day1, and the medium was replaced with the normal medium again on day3. Thereafter, the medium was changed every two days, and the cells were collected on day 10 (laminin group). As a control, cells cultured under the same conditions using a dish without laminin coat were collected (control group).

Total RNA was prepared from the obtained cells, and cDNA was obtained by reverse transcription reaction. By using the obtained cDNA as a template, each of Fabp4, PPARγ, GLUT4, LPL, ACC-1, FAS, DGAT, HSL and ATGL by quantitative PCR using 7500 Fast Real-Time PCR System (Applied Biosystems) The expression level of the gene was measured.
Fabp4 is known as a differentiation / maturation marker for adipocytes. PPARγ is known as a master regulator of adipocyte differentiation. GLUT4 and LPL are genes related to sugar and lipid uptake, ACC-1, FAS and DGAT are lipid synthesis-related genes, HSL and ATGL are lipolysis-related genes, both of which are involved in energy metabolism. Known as the major gene involved.
The measured expression level of each gene was corrected by an internal standard (36B4 gene expression level). The obtained values were graphed as an average value ± standard error (n = 3). The significant difference test was performed using Student's t-test.

  The results are shown in FIG. The level of expression of Fabp4, an adipocyte differentiation / maturation marker, was significantly increased by laminin and reached 2.4 times that of the control group. In addition, the mRNA expression level of the transcription factor PPARγ, which is a master regulator of adipocyte differentiation, was significantly increased by laminin and reached 3.5 times that of the control group. Furthermore, the expression of major genes involved in energy metabolism (GLUT4, LPL, ACC-1, FAS and DGAT) was also significantly increased by laminin. From these results, it was shown that laminin promotes differentiation and maturation of adipocytes. Furthermore, GLUT4, LPL, ACC-1, FAS and DGAT are genes involved in sugar / lipid uptake and lipid synthesis, indicating that laminin promotes fat accumulation in adipocytes. Therefore, suppression of laminin signal leads to suppression of adipocyte differentiation and maturation and fat accumulation, and laminin signal is effective as an index for searching for adipocyte differentiation maturation inhibitor or fat accumulation inhibitor. .

Reference Example 2 Effect of promoting fat accumulation by laminin After fixing fat cells collected by the same method as in Example 1 with 10% formalin, lipid droplets in the cells were stained with Oil-red O staining solution, and examined with a stereomicroscope. Observed and photographed. The Oil-red O staining solution used was an Oil red-O stock solution (0.5% Oil red-O / isopropanol) and DDW mixed at 3: 2 and filtered through a 0.5 mm filter. In order to quantify the degree of staining (fat content) of the cells, the pigment was extracted from the cells with isopropanol, the absorbance OD450 was measured, and the relative absorbance with the absorbance of the control group being 1 was determined. The obtained absorbance was graphed as an average value ± standard error (n = 3). The significant difference test was performed using Student's t-test.

  The results are shown in FIG. The oil-red O staining degree of the laminin group was stronger than that of the control group (FIG. 2A), and a significant difference was also observed in the quantitative analysis of the staining degree (FIG. 2B). These results indicated that laminin promoted fat cell fat accumulation. Accordingly, suppression of laminin signal leads to suppression of fat accumulation in adipocytes, and laminin signal is effective as an index for searching for a fat cell fat accumulation inhibitor.

Example 1 Effect of suppressing differentiation / maturation of adipocytes by suppressing laminin signal Laminin partial peptide YIGSR (Tyr-Ile-Gly-Ser-Arg: SEQ ID NO: 1) is a partial peptide of laminin β1 chain. This peptide is the major receptor binding sequence of laminin and competitively inhibits laminin from binding to laminin receptor (Graf et al, Biochemistry, 1987, 26 (22): 6896-6900, and Nomizu) Basics, Protein, Nucleic Acid, Enzyme, 2000, 45 (14): 2475-2482). The effect of inhibiting differentiation and maturation of adipocytes by this laminin partial peptide was examined.

  C-terminal amidated peptide in which the C-terminal of the following five peptides (SEQ ID NO: 1 to 5) including the amino acid sequence shown in SEQ ID NO: 1 and having different chain lengths are amidated, and non-amidated shown in SEQ ID NO: 1 Peptides were purchased from Biomatik Corporation. These six peptides (Table 1 and FIG. 3) were used in the following experiments.

Primary cultured cells derived from rat subcutaneous fat were suspended in a normal medium supplemented with any of test peptides 1 to 6 (final concentration 100 μg / ml), incubated at 37 ° C. for 30 minutes, and then laminin-coated 6-well dish (BD company). Thereafter, any of the above test peptides 1 to 6 (final concentration 100 μg / ml) was added to all the culture media.
Cells are normally cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum under conditions of 37 ° C and 5% CO ₂ until they become 100% confluent, then the medium is replaced with a differentiation-inducing medium. did. The differentiation induction medium used was a medium supplemented with 5 μg / ml insulin, 1 μM dexamethasone, and 0.115 mg / ml isobutylmethylxanthine. The day when the medium was changed to the differentiation-inducing medium was defined as day1, and the medium was replaced with the normal medium again on day3. Thereafter, the medium was changed every two days, and the cells were collected on day 10 (laminin group). As a control, cells cultured under the same conditions using a dish without laminin coat were collected (control group).
Total RNA was prepared from the obtained cells, and quantitative PCR was performed on the expression of Fabp4, which is known as a differentiation / maturation marker for adipocytes, using cDNA obtained by reverse transcription as a template. The Fabp4 gene expression level was corrected by an internal standard (36B4 gene expression level). The obtained values were graphed as an average value ± standard error (n = 3). The significant difference test was performed using Student's t-test.

  The results are shown in FIG. The expression of Fabp4, which is a differentiation / maturation marker of adipocytes, increases in the presence of laminin (laminin coat group), but when a C-terminal amidated form of the laminin partial peptide represented by SEQ ID NO: 1 (test peptide 1) is added, laminin And the binding of the receptor to the receptor were competitively inhibited, and the expression level of Fabp4 was significantly reduced (repression rate 44%). In addition, the laminin partial peptide (test peptide 2) represented by SEQ ID NO: 2 also showed a tendency to decrease the Fabp4 expression level (suppression rate 14%). On the other hand, when a laminin partial peptide represented by SEQ ID NO: 1 (test peptide 6) that does not amidate the C-terminus was added, no decrease in Fabp4 expression was observed. The C-terminal amidated form of the laminin partial peptide represented by SEQ ID NOs: 1 and 2 can suppress the progress of the differentiation and maturation process of adipocytes caused by laminin, and can suppress fat accumulation by mature adipocytes.

Production Example Production Example 1 Tablet A composition having the following composition was tableted to produce a tablet (1 tablet = 250 mg).

Composition Blending amount (% by mass)
Polypeptide (SEQ ID NO: 1) 10
Cornstarch 50
Cellulose 10
Lactose 30

Production Example 2 Capsule A composition having the following composition was encapsulated in a capsule to produce a capsule (1 capsule = 300 mg).

Composition Blending amount (% by mass)
Polypeptide (SEQ ID NO: 1) 15
Vitamin C 20
Cellulose 10
Cornstarch 40
Tocopherol 2
Lactose 13

Production Example 3 External Preparation Raw materials having the following composition were mixed by a conventional method to produce an external preparation.

Composition Blending amount (% by mass)
Polypeptide (SEQ ID NO: 1) 0.5
Glycerol monostearate 1
Ethanol 15
Propylene glycol 4
Isopropyl palmitate 3
Lanolin 1
Methyl paraoxybenzoate 0.1
Fragrance, pigment Trace amount Purified water Remaining

Production Example 4 Injection A raw material having the following composition was mixed by a conventional method and sterilized by heating to produce an injection (1 ampoule = 5 mL).

Composition Blending amount Polypeptide (SEQ ID NO: 1) 15 mg
Glucose 100mg
5mL distilled water for injection

Claims (8)

  An adipocyte differentiation and maturation inhibitor comprising as an active ingredient a C-terminal amidated product of a polypeptide comprising an amino acid sequence having 0 to 3 amino acids bonded to the N-terminal side of the amino acid sequence represented by SEQ ID NO: 1 and / or Fat accumulation inhibitor.   The adipocyte differentiation / maturation inhibitor and / or fat accumulation inhibitor according to claim 1, wherein the polypeptide consists of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.   The adipocyte differentiation / maturation inhibitor and / or fat accumulation inhibitor according to claim 1 or 2, wherein the polypeptide is bound to polyethylene glycol.   An obesity-preventing and / or improving agent comprising, as an active ingredient, a C-terminal amidated product of a polypeptide comprising an amino acid sequence in which 0 to 3 amino acids are bonded to the N-terminal side of the amino acid sequence represented by SEQ ID NO: 1.   The agent for preventing and / or improving obesity according to claim 4, wherein the polypeptide consists of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.   The obesity preventing and / or ameliorating agent according to claim 4 or 5, wherein the polypeptide is bound to polyethylene glycol.   A pharmaceutical or quasi-drug which comprises as an active ingredient a C-terminal amidated product of a polypeptide comprising an amino acid sequence in which 0 to 3 amino acids are bonded to the N-terminal side of the amino acid sequence represented by SEQ ID NO: 1.   An external preparation comprising as an active ingredient a C-terminal amidated product of a polypeptide comprising an amino acid sequence having 0 to 3 amino acids bonded to the N-terminal side of the amino acid sequence represented by SEQ ID NO: 1.