JP2005213206A - Agent for ameliorating carbohydrate metabolizing function - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a new agent for ameliorating a carbohydrate-metabolizing function, more precisely, an agent for treating or preventing diseases associated with the carbohydrate metabolism and having activities for promoting the transmittance of insulin signals. <P>SOLUTION: This agent for treating or preventing the diseases associated with the carbohydrate metabolism contains a protein having one of the following amino acid sequences or a fragment thereof as an active ingredient: (a) a specific amino acid sequence, (b) an amino acid sequence obtained by deleting, adding or substituting one or several amino acids from, to or for the amino acid sequence of (a), (c) a partial amino acid sequence obtained by deleting 26 amino acids from the amino terminus of the amino acid sequence of (a), (d) an amino acid sequence obtained by adding methionine to the amino terminus of the amino acid sequence of (c), (e) an amino acid sequence having ≥80% sequence identity with the amino acid sequence of (a), or the like. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a glucose metabolism function improving agent, specifically, a therapeutic agent for a glucose metabolism-related disease characterized by having an insulin signal transduction promoting activity such as a sugar uptake promoting activity in muscle or a gluconeogenesis inhibiting activity in liver. It relates to preventive agents.

Diabetes is a condition in which the homeostasis of glucose metabolism (sugar renewal and clearance) in the living body is broken, particularly a hyperglycemic condition. In the background of the pathology of diabetes, insulin secretion, which is a glycemic hormone, is dysfunctional or dysfunctional. By correcting or substituting insulin action, treatment of diseases closely related to glucose metabolism (sugar metabolism-related diseases) or Prevention is possible. So far, insulin has been known as an in vivo substance (hormone) for improving the sugar metabolism function, and insulin preparations have been used for improving the sugar metabolism function. However, more excellent drugs are required.
On the other hand, the protein consisting of the amino acid sequence represented by SEQ ID NO: 2 is used for diagnosis of fat accumulation syndrome (see Patent Document 1) and has a fat accumulation promoting action in fat cells (see Patent Document 2). Etc. are known. However, the insulin-like activity of the protein has not been known.
International Publication No. 00/62073 Pamphlet International Publication No. 02/10772 Pamphlet

  The problem to be solved by the present invention is to provide a novel sugar metabolism function improving agent, specifically, a therapeutic or prophylactic agent for a sugar metabolism-related disease, characterized by having insulin signal transduction promoting activity.

As a result of intensive studies under these circumstances, the present inventors have found that the protein consisting of the amino acid sequence represented by SEQ ID NO: 2 has an activity of improving the glucose metabolism function, specifically, an insulin signaling promoting activity, Thus, the present inventors have found that it exhibits an action to improve the sugar metabolism function, and have completed the present invention.
That is, the present invention

[1] Glucose metabolism containing a protein comprising any one of the following amino acid sequences (a) to (h) (hereinafter sometimes referred to as the present protein in the present specification) or a fragment thereof as an active ingredient Therapeutic or prophylactic agent for related diseases;
<Amino acid sequence group>
(A) the amino acid sequence represented by SEQ ID NO: 2,
(B) an amino acid sequence in which one or more amino acids are deleted, added or substituted in the amino acid sequence represented by SEQ ID NO: 2;
(C) a partial amino acid sequence obtained by deleting 26 amino acids from the amino terminus in the amino acid sequence represented by SEQ ID NO: 2;
(D) an amino acid sequence in which methionine is added to the amino terminus of the amino acid sequence of (c),
(E) an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2,
(F) an amino acid sequence encoded by DNA having a base sequence represented by nucleotides from the 18th nucleotide to the 1493th nucleotide in the base sequence represented by SEQ ID NO: 1;
(G) It is encoded by DNA having a base sequence having 80% or more sequence identity with DNA having the base sequence shown by nucleotides 18 to 1493 in the base sequence shown by SEQ ID NO: 1. And an amino acid sequence having insulin-like activity,
(H) Hybridizes under stringent conditions with DNA having complementarity to DNA having the nucleotide sequence represented by nucleotides from the 18th nucleotide to the 1493th nucleotide in the nucleotide sequence represented by SEQ ID NO: 1. An amino acid sequence encoded by DNA and having insulin-like activity [2] The therapeutic or prophylactic agent according to [1], which enhances insulin-like activity;
[3] The therapeutic or prophylactic agent according to [2], wherein the insulin-like activity is an insulin signal transduction promoting activity in hepatocytes or muscle cells;
[4] The therapeutic or preventive agent according to [2], wherein the insulin-like activity is a gluconeogenesis-inhibiting activity in hepatocytes or a glucose uptake promoting activity in muscle cells;
[5] The therapeutic or prophylactic agent according to [2], wherein the insulin-like activity is a hypoglycemic effect;
[6] A method for assaying insulin-like activity,
(1) a first step of bringing a test substance into contact with muscle cells or hepatocytes,
(2) a second step of measuring insulin-like activity in the cell, and (3) the first step and the second step using the present protein instead of the test substance as the measurement value measured in the second step. A third step of evaluating the insulin-like activity of the test substance using as an index that the insulin-like activity of the test substance is equal to or greater than the insulin-like activity of the protein,
A method for assaying insulin-like activity, comprising:
[7] The assay method according to [6], wherein the insulin-like activity is insulin signal transduction promoting activity;
[8] The assay method according to [7], wherein the insulin signaling promoting activity is measured using the amount of phosphorylated insulin receptor, IRS-1, PI3 kinase or Akt as an index;
[9] The assay method according to [6], wherein the insulin-like activity is gluconeogenesis-inhibiting activity in hepatocytes;
[10] The assay method according to [6], wherein the insulin-like activity is an activity of promoting sugar uptake into muscle cells;
[11] The assay method according to any one of [6], [7], [8] and [10], wherein the muscle cell used in the first step is a skeletal muscle cell;
[12] The assay method according to any one of [6] to [9], wherein the hepatocytes used in the first step are established hepatocytes or primary cultured hepatocytes;
[13] Insulin-like activity of a test substance using as an index whether or not the test substance promotes the expression of the present protein or a gene encoding the present protein (hereinafter sometimes referred to as the present gene) The testing method of
[14] The method for assaying insulin-like activity according to [13], comprising the following steps (1) to (3):
(1) a first step of bringing a test substance into contact with a cell capable of expressing a gene encoding the protein according to [13],
(2) a second step of measuring an expression level of the gene in a cell contacted with a test substance and comparing the expression level with an expression level of the gene in a control cell not contacted with the test substance; and (3) A third step of evaluating the insulin-like activity of the test substance using as an index whether or not the test substance increases the expression of the gene based on the comparison result of the second step;
[15] The method for assaying insulin-like activity according to [13], comprising the following steps (1) to (3):
(1) a first step of contacting a test substance with a cell capable of expressing the protein according to [13],
(2) a second step of measuring the expression level of the protein in a cell contacted with a test substance, and comparing the expression level with the expression level of the protein in a control cell not contacted with the test substance; A third step of evaluating the insulin-like activity of the test substance using as an index whether or not the test substance increases the expression of the protein based on the comparison result of the second step;
[16] The method for assaying insulin-like activity according to [13], comprising the following steps (1) to (3):
(1) a first step of bringing a test substance into contact with a cell containing a reporter gene operably linked to an expression control region of the gene encoding the protein according to [13],
(2) a second step of measuring the expression level of the reporter gene in a cell contacted with a test substance, and comparing the expression level with the expression level of the gene in a control cell not contacted with the test substance; and (3) A third step of evaluating the insulin-like activity of the test substance using as an index whether or not to increase the expression level of the reporter gene based on the comparison result of the second step;
[17] The assay method according to any one of [13] to [16], wherein the insulin-like activity is insulin signal transduction promoting activity;
[18] The assay method according to any one of [13] to [16], wherein the insulin-like activity is an activity to promote sugar uptake into muscle cells;
[19] The assay method according to any one of [13] to [16], wherein the insulin-like activity is gluconeogenesis-inhibiting activity in hepatocytes;
[20] Selecting a candidate substance for a therapeutic or prophylactic agent for a glucose metabolism-related disease using the insulin-like activity of the test substance as an index measured by the assay method according to any one of [6] to [19] A method for searching for a therapeutic or prophylactic agent for a glucose metabolism-related disease,
[21] The search method according to [20], wherein the therapeutic or preventive agent for a glucose metabolism-related disease is an insulin signaling promoter in muscle cells or hepatocytes;
[22] The searching method according to [20], wherein the therapeutic or preventive agent for a sugar metabolism-related disease is a sugar uptake promoter in muscle cells;
[23] The search method according to [20], wherein the therapeutic or preventive agent for a glucose metabolism-related disease is a gluconeogenesis inhibitor in the liver;
[24] A therapeutic or preventive agent for a sugar metabolism-related disease, containing as an active ingredient a substance selected by the search method according to any one of [20] to [23];
About.

  According to the present invention, there is used a therapeutic or preventive agent for a sugar metabolism-related disease characterized by having an activity of improving glucose metabolism function, specifically, an insulin signaling promoting activity, an assay method for insulin-like activity, and the assay method It has become possible to provide a method for searching for a therapeutic or preventive agent for a sugar metabolism-related disease.

The present invention is described in detail below.
The first aspect of the present invention relates to a therapeutic or prophylactic agent for a sugar metabolism-related disease containing the protein or a fragment thereof as an active ingredient.
In the present specification, “a protein comprising any one of the following amino acid sequences (a) to (h)” (the present protein) means (a) an amino acid sequence represented by SEQ ID NO: 2 and (b) SEQ ID NO: 2 (C) In the amino acid sequence shown in SEQ ID NO: 2, 26 amino acids were deleted from the amino terminus thereof. (D) an amino acid sequence in which methionine is added to the amino terminus of the amino acid sequence of (c), (e) an amino acid having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2 (F) having a base sequence represented by nucleotides from the 18th nucleotide to the 1493th nucleotide in the base sequence represented by SEQ ID NO: 1 (G) 80% or more of sequence identity with the DNA having the base sequence represented by nucleotides 18 to 1493 in the base sequence represented by SEQ ID NO: 1 Complementary to an amino acid sequence encoded by a DNA having a nucleotide sequence having the nucleotide sequence and (h) a DNA having a nucleotide sequence represented by nucleotides from the 18th nucleotide to the 1493th nucleotide in the nucleotide sequence represented by SEQ ID NO: 1. And a protein having an insulin-like activity and comprising an amino acid sequence encoded by a DNA that hybridizes under stringent conditions.

Here, the “amino acid deletion, addition or substitution” in (b) and “80% or more sequence identity” in (e) and (g) include, for example, the amino acid represented by SEQ ID NO: 2 This includes a process that a protein having a sequence undergoes in a cell, a naturally occurring mutation due to a species difference, an individual difference, a difference between tissues, etc. from which the protein is derived, and an artificial amino acid mutation.
As a technique for artificially performing the “amino acid deletion, addition or substitution” in the above (b) (hereinafter sometimes referred to as amino acid modification as a whole), for example, the amino acid sequence represented by SEQ ID NO: 2 is used. There is a technique in which conventional site-directed mutagenesis is performed on DNA encoding, and then this DNA is expressed by a conventional method. Here, as a site-specific mutagenesis method, for example, a method using amber mutation (gapped duplex method, Nucleic Acids Res., 12,9441-9456 (1984)), a PCR method using a mutagenesis primer Etc.
The number of amino acids modified as described above is at least one residue, specifically one or several, or more. The number of such modifications may be in a range where the insulin-like activity of the protein can be found.
Of the deletions, additions or substitutions, the modification relating to amino acid substitution is particularly preferable. The substitution is more preferably substitution with an amino acid having similar properties such as hydrophobicity, charge, pK, and structural features. Examples of such substitution include (1) glycine, alanine; (2) valine, isoleucine, leucine; (3) aspartic acid, glutamic acid, asparagine, glutamine, (4) serine, threonine; (5) lysine, arginine. And (6) substitution within the group of phenylalanine and tyrosine.

In the present invention, “sequence identity” refers to sequence identity and homology between two DNAs or two proteins. The “sequence identity” is determined by comparing two sequences that are optimally aligned over the region of the sequence to be compared. Here, the DNA or protein to be compared may have an addition or a deletion (for example, a gap) in the optimal alignment of the two sequences. Such sequence identity can be calculated, for example, by creating an alignment using the ClustalW algorithm (Nucleic Acid Res., 22 (22): 4673-4680 (1994)) using Vector NTI. The sequence identity is measured using sequence analysis software, specifically, an analysis tool provided by Vector NTI, GENETYX-MAC, or a public database, for example, the homepage address http It is generally available at: //www.ddbj.nig.ac.jp.
The sequence identity in the present invention may be 80% or more, preferably 90% or more, more preferably 95% or more.

  Regarding “hybridize under stringent conditions” in (h) above, the hybridization used here is, for example, by Sambrook J., Frisch EF, Maniatis T., Molecular Cloning 2nd Edition (Molecular Cloning 2nd edition), Cold Spring Harbor Laboratory press, etc. (Cold Spring Harbor Laboratory press). “Stringent conditions” means, for example, 6 × SSC (a solution containing 1.5M NaCl, 0.15M trisodium citrate is 10 × SSC), 45% in a solution containing 50% formamide. The conditions (Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6), etc., where a hybrid is formed at 50 ° C. and then washed with 2 × SSC at 50 ° C. it can. The salt concentration in the washing step can be selected, for example, from 2 × SSC at 50 ° C. (low stringency conditions) to 0.2 × SSC at 50 ° C. (high stringency conditions). The temperature in the washing step can be selected, for example, from room temperature (low stringency conditions) to 65 ° C. (high stringency conditions). It is also possible to change both the salt concentration and the temperature.

  In the present specification, the fragment of the protein represents a peptide fragment consisting of an amino acid partial sequence of 15 to 100 residues, preferably 15 to 50 residues of the protein, and retains the insulin-like activity of the protein. As long as there is no particular limitation.

The method for preparing this protein will be described below.
First, a gene encoding this protein (this gene), for example, (I) a base sequence encoding the amino acid sequence represented by SEQ ID NO: 2, and (II) one or more amino acids in the amino acid sequence represented by SEQ ID NO: 2 Is a base sequence encoding an amino acid sequence deleted, added or substituted, (III) a base sequence encoding a partial amino acid sequence in which 26 amino acids have been deleted from the amino terminus in the amino acid sequence shown in SEQ ID NO: 2 A sequence (that is, a base sequence encoding an amino acid sequence represented by amino acids 27 to 491 in the amino acid sequence represented by SEQ ID NO: 2), (IV) in the amino acid sequence of (III), the amino terminal (V) the amino acid sequence represented by SEQ ID NO: 2 and 80 A nucleotide sequence encoding the amino acid sequence having the above sequence identity, (VI) a nucleotide sequence represented by SEQ ID NO: 1, and (VII) a nucleotide sequence from the 18th nucleotide to the 1493th nucleotide in the nucleotide sequence represented by SEQ ID NO: 1. (VIII) a nucleotide having a sequence identity of 80% or more with a DNA having a nucleotide sequence represented by nucleotides from the 18th nucleotide to the 1493th nucleotide in the nucleotide sequence represented by SEQ ID NO: 1 A DNA having a complementarity to a DNA having a nucleotide sequence represented by a nucleotide sequence from the 18th nucleotide to the 1493th nucleotide in the sequence or (IX) the nucleotide sequence represented by SEQ ID NO: 1, and under stringent conditions A gene having a base sequence such as a base sequence of DNA to be hybridized is converted into a conventional genetic engineering method (for example, Acquired according to Sambrook J., Frisch EF, Maniatis T., methods described in Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory press). Next, by using the obtained present gene, the present protein is produced and obtained according to an ordinary genetic engineering method. In this way, the present protein can be prepared.

  For example, a culture obtained by preparing a plasmid capable of expressing this gene in a host cell, introducing it into the host cell, transforming it, and then culturing the transformed host cell (transformant). What is necessary is just to acquire this protein from a thing. Examples of the plasmid include a promoter that contains genetic information that can be replicated in a host cell, can be propagated autonomously, can be easily isolated and purified from the host cell, and can function in the host cell. Preferred examples include those in which a gene encoding the present protein is introduced into an expression vector having a detectable marker. Various types of expression vectors are commercially available. For example, an expression vector used for expression in E. coli is an expression vector containing a promoter such as lac, trp, tac, etc., and these are commercially available from Pharmacia, Takara Shuzo and others. Restriction enzymes used for introducing a gene encoding this protein into the expression vector are also commercially available from Takara Shuzo. If it is necessary to induce higher expression, a ribosome binding region may be linked upstream of the gene encoding this protein. Examples of the ribosome binding region used include those described in reports by Guarente L. et al. (Cell 20, p543) and Taniguchi et al. (Genetics of Industrial Microorganisms, p202, Kodansha).

  The vector used for expression in mammalian cells is an expression vector containing a promoter such as SV40 virus promoter, cytomegalovirus promoter (CMV promoter), Raus Sarcoma Virus promoter (RSV promoter), β-actin gene promoter, aP2 gene promoter, etc. These are commercially available from Toyobo and Takara Shuzo.

Examples of host cells include prokaryotic or eukaryotic microbial cells, insect cells, or mammalian cells. For example, Escherichia coli and the like can be preferably mentioned from the viewpoint of easy mass preparation of the present protein.
The plasmid obtained as described above can be introduced into the host cell by an ordinary genetic engineering method.

The transformant can be cultured by a conventional method used for culturing microorganisms, insect cells or mammalian cells. For example, in the case of Escherichia coli, culturing is performed in a medium appropriately containing an appropriate carbon source, nitrogen source, and micronutrients such as vitamins. The culture method may be any of solid culture and liquid culture, and preferred examples include liquid culture such as aeration and agitation culture.

  Acquisition of this protein may be carried out by combining methods commonly used for isolation and purification of general proteins. For example, the transformants obtained by the above culture are collected by centrifugation or the like, and the transformants are crushed or dissolved, and if necessary, proteins are solubilized, and various types such as ion exchange, hydrophobicity, gel filtration, etc. What is necessary is just to refine | purify the process using a chromatography individually or in combination. An operation of restoring the higher order structure of the purified protein may be further performed. For example, the transformant obtained by the above culture may be removed by centrifugation or the like, and the present protein may be purified from the culture supernatant in the same manner as described above.

In the present specification, examples of the glucose metabolism-related diseases include diseases associated with abnormal glucose metabolism functions in the living body. Specifically, diseases such as diabetes, impaired glucose tolerance, arteriosclerosis associated with sustained hyperglycemia, and the like. Can be mentioned. That is, the sugar metabolism-related disease is not particularly limited as long as it is a disease having various pathologies caused by a decrease in insulin function or dysfunction. As a disease to which the therapeutic or preventive agent for a sugar metabolism-related disease of the present invention can be applied, a disease state in which blood glucose homeostasis such as diabetes is broken can be mentioned.
As used herein, “insulin-like activity” refers to a physiological action that occurs when insulin binds to an insulin receptor, and specifically includes a hypoglycemic action, a gluconeogenesis-inhibiting activity in the liver, or a sugar in muscle cells. Examples thereof include uptake promoting activity.
When insulin binds to the insulin receptor, the β subunit, which is the intracellular domain of the insulin receptor, is autophosphorylated, and insulin-derived signal transduction occurs in the cell (hereinafter referred to as insulin signal transduction). Specific examples of insulin signaling include phosphorylation of insulin receptor, IRS-1, PI3 kinase, or Akt.
“Insulin signal transduction promoting activity” refers to the activity of enhancing the above-mentioned insulin signal transduction, specifically, enhancing the phosphorylation of insulin receptor, IRS-1, PI3 kinase or Akt in hepatocytes or muscle cells. Activity, that is, the activity of increasing the phosphorylated form of insulin receptor, IRS-1, PI3 kinase or Akt.

  “Glucogenesis in hepatocytes” refers to glucose biosynthesis in the liver, and represents intracellular metabolism in which glucose is biosynthesized from pyruvate via oxaloacetate. It is known that the rate-limiting step of gluconeogenesis is a reaction catalyzed by phosphoenolpyruvate carboxylase (PEPCK), and insulin is known to exhibit gluconeogenesis-inhibiting activity by suppressing the expression level of PEPCK. ing.

  “Sugar uptake promoting activity in muscle cells” refers to the activity of promoting the uptake of glucose from outside the cell via the glucose transporter. Insulin is known to promote glucose uptake by translocating GLUT4 stored in the cell onto the cell membrane. The taken-in glucose is converted into pyruvic acid through a glycolysis system, and at the same time, ATP is produced and used as energy.

  The therapeutic or preventive agent for a sugar metabolism-related disease of the present invention is characterized by enhancing insulin-like activity. Here, specifically, insulin-like activity is enhanced by 1) suppressing gluconeogenesis in the liver, 2) promoting glucose uptake in muscle cells, 3) promoting insulin signaling, 4 ) Represents a decrease in blood sugar level.

A second aspect of the present invention is a method for assaying insulin-like activity, comprising:
(1) a first step of bringing a test substance into contact with muscle cells or hepatocytes,
(2) a second step of measuring insulin-like activity in the cell, and (3) the first step and the second step using the present protein instead of the test substance as the measurement value measured in the second step. A third step of evaluating the insulin-like activity of the test substance using as an index that the insulin-like activity of the test substance is equal to or greater than the insulin-like activity of the protein,
Have

The muscle cells used in the first step include muscle cells isolated from animal muscle tissues and muscle cells (muscle tissues) forming a group having the same function and form. There is no particular limitation as long as it is a cell that expresses the body, exhibits an activity of promoting glucose uptake in response to insulin, and functions in signal transduction downstream of insulin, and may be a cell in any differentiation process. Examples include myoblasts and cells that have differentiated to form myotubes. Preferably, differentiated muscle cells are used. Here, the myoblast refers to an undifferentiated cell that has acquired a base as a muscle cell, and is in a state that is not multinucleated or filamentized as after differentiation. Differentiated muscle cells are usually multinuclear cylindrical cells with a diameter of 10 to 100 μm and up to 30 cm, and have the ability to take up sugar.
Here, examples of the “muscle tissue” include skeletal muscle tissues such as soleus muscle or extensor digitorium longus.
Examples of the muscle cell-derived animal include mammals, and more specifically human, monkey, mouse, rat, hamster and the like.

  Muscle cells can be isolated from skeletal muscle tissue, but established cells such as L6 (Dainippon Pharmaceutical) and C2C12 can also be used. These are commercially available or can be easily obtained from cell deposit institutions such as RIKEN Cell Bank. For example, fetal bovine serum (hereinafter referred to as FBS) (GIBCO), penicillin, and streptomycin (GIBCO) are added to these cells to a final concentration of 10%, 100 units / ml, and 100 μg / ml, respectively. Can be cultured in a normal medium such as Dulbecco's modified Eagle medium (containing 4.5 g / L D-glucose and 584 mg / L L-glutamine; manufactured by GIBCO) (hereinafter referred to as DMEM medium). After becoming confluent, they differentiate into cylindrical multinucleated muscle cells.

  The hepatocytes used in the first step express hepatocytes in which enzymes involved in gluconeogenesis and glycolysis function, that is, express insulin receptor, and suppress gluconeogenesis in response to insulin. Any hepatocyte may be used as long as it is a cell, and hepatocytes isolated from the liver of an animal, and hepatocytes (liver tissue) forming a population having the same function / morphology are included. Examples of animal movements derived from the hepatocytes include mammals, and more specifically humans, monkeys, mice, rats, hamsters and the like.

Examples of hepatocytes include primary cultured hepatocytes, which can be prepared by the following method. That is, rats (Wister rat, 150-250g) are incised in the abdomen, the intestine is moved to the side, the portal vein is exposed, a 22G indwelling needle (Terumo) is inserted and fixed with a suture. The infusion tube is connected to the tube of the peristaltic pump, the indwelling needle and the tube are connected so that air does not enter while the perfusate (Liver Perfusion Medium) (Gibco) is allowed to flow out. Start perfusion with the perfusate kept in place. After perfusion for 10 minutes, perfuse the collagenase perfusion solution (Liver Digest Medium) (Gibco) maintained at 37 ° C in a constant temperature layer for 10 minutes. After completion of perfusion, the liver was removed, mince on a petri dish, suspended in Hepatocyte Wash Medium (Gibco) or Medium 199 (Gibco) containing 5% FBS, filtered through a 250 μm mesh filter, Centrifuge at 500 rpm for 1 min. Remove the supernatant. The precipitate is suspended in the washing medium, centrifuged, and the supernatant removed three more times, filtered through a 100 μm cell strainer (FALCON), suspended in William's Medium E (Sigma) containing 10% FBS, and centrifuged. Remove the supernatant. Finally, the precipitate is suspended in William's Medium E containing 10% FBS, 1 nM insulin (manufactured by Sigma) and 100 nM dexamethasone (manufactured by Sigma). Measure and spread on a collagen-coated plate (Iwaki) at a density of 0.5 to 1.5 × 10 ⁵ cells / cm ² . Replace the medium with the same medium 2-3 hours after seeding. The primary cultured hepatocytes thus prepared can be used for the assay from the next day.

  In addition, established cells such as H4IIE and HepG2 can be used, and these are commercially available from Dainippon Pharmaceutical Co., Ltd. and can be easily obtained. These hepatocytes can be cultured by a normal cell culture method. For example, a medium in which dexamethasone (Sigma) and cAMP (Sigma) are added to DMEM medium so as to have final concentrations of 100 nM and 50 μM, respectively (hereinafter referred to as “DMEM”). , Medium for hepatocytes).

  The compound used as the test substance is not particularly limited, and examples thereof include proteins, peptides, nucleic acids, inorganic compounds, natural or synthetically prepared organic compounds, and the like. The test substance is specifically a peptide library of 3 to 50 amino acids, preferably 5 to 20 amino acids, or a molecular weight of 100 to 2000, preferably prepared using combinatorial chemistry techniques known to those skilled in the art. Can include 200 to 800 low molecular weight organic compound libraries.

  The concentration of the test substance to be brought into contact with muscle cells or hepatocytes is not particularly limited, and is usually about 0.1 μM to about 100 μM, preferably 1 μM to 50 μM. The time for contacting the muscle cell or hepatocyte with the test substance is usually about 5 minutes to 30 minutes, preferably about 10 minutes to 20 minutes. The test substance can be used by appropriately dissolving or suspending in a solvent such as water, a buffer such as phosphate buffer or Tris buffer, ethanol, acetone, dimethyl sulfoxide, or a mixture thereof.

In the second step, methods for measuring insulin-like activity include the following 1) to 3):
1) Activity of promoting sugar uptake in muscle cells or muscle tissue Any method for measuring the amount of sugar taken up in muscle cells or muscle tissue can be used as long as it is a method for measuring the amount of sugar taken up in muscle cells or muscle tissue. For example, a method of quantifying incorporated radiolabeled glucose can be used. Specific examples of a method for quantifying radiolabeled glucose incorporated into muscle tissue or cells include: (1) The amount of radiolabeled glucose incorporated into tissue using muscle tissue isolated from a living body Examples include a method of measuring, and (2) a method of measuring the amount of radiolabeled glucose incorporated into the cells using differentiated muscle cells. As a method for measuring sugar uptake using muscle tissue (the former), for example, the method described in Molecular Cell, vol. 2, p. 559 (1998) can be used. Examples of a method for measuring the amount of radiolabeled glucose incorporated into the cells using differentiated muscle cells (the latter) include “Am. J. Physiol., Vol. 276, E849 (1999)” and “ The method described in “History of Medicine, 184 (6), 503-506, 1998” can be used.

2) Glucogenesis inhibitory activity in hepatocytes As a method for measuring the amount of gluconeogenesis in hepatocytes, for example, a method described in the literature or the like can be used. More specifically, as described above, H4IIE cells cultured in a hepatocyte culture medium are seeded in a 12-well plate in an amount of about 3 × 10 ⁵ cells per well, and the cells become confluent and contain 5 mM glucose. Change to the same medium for hepatocytes and culture overnight. After washing the cells with phosphate buffer, gluconeogenesis buffer (8.3 g / L DMEM-base, 4 mM L-glutamine, 1 mM sodium pyruvate, 3.7 g / L sodium bicarbonate, 0.9 g / L lactic acid, 100 units / mL penicillin and 100 μg / mL streptomycin) and further culture the cells overnight. The next day, the cell supernatant is collected, diluted as appropriate, and the amount of sugar (glucose) contained in the supernatant is quantified. Many kits for determining glucose are commercially available. For example, F-kit glucose (JK International) can be used.

3) Insulin signal transduction promoting activity in muscle cells or hepatocytes Here, methods for measuring insulin signaling in muscle cells or hepatocytes include insulin receptor and its downstream molecules, IRS-1 protein, PI3 kinase, Akt. Examples thereof include a method for quantifying the amount of phosphorylation of a protein. Here, IRS-1 protein, PI3 kinase, and Akt protein are known, and their structures and functions are described in Molecular Medicine Vol. 36, 110-115 (1999; Nakayama Shoten). Examples of a method for quantifying the phosphorylation amount of these molecules include Western blotting using an antibody. Antibodies used in the method are commercially available from, for example, Sigma, Upstate Biotechnology, etc. It can be performed according to.

In the third step of the present invention, “the measured value when the first step and the second step are performed using the present protein instead of the test substance” means (i) muscle cells or hepatocytes, and sequence This represents a measured value obtained by contacting the present protein such as a protein comprising the amino acid sequence represented by No. 2 and (ii) measuring insulin-like activity in the cell.
Here, the concentration of the protein to be contacted with muscle cells or hepatocytes is usually about 0.1 ng / ml (2 pM) to about 100 μg / ml (2 μM), and about 1 ng / ml (20 pM) to about 20 μg. / Ml (0.4 μM) is preferred. The time for contacting the muscle cells or hepatocytes with this protein is usually 5 minutes to 1 day, preferably about 5 minutes to 30 minutes. The concentration of the test substance to be contacted with muscle cells or hepatocytes is usually about 0.1 μM to about 100 μM, and preferably 1 μM to 50 μM. The time for contacting the muscle cells or hepatocytes with the test substance is usually about 5 minutes to 1 day, preferably about 5 minutes to 30 minutes, and more preferably about 10 minutes to 20 minutes.

  The first step and the second step in the case of using the measured value measured in the second step of the present invention and the present protein such as the protein consisting of the amino acid sequence represented by SEQ ID NO: 2 instead of the test substance were performed. By comparing the measured values, the test substance exhibiting insulin-like activity equivalent to or higher than that of the present protein can be evaluated as a substance having insulin-like activity.

In addition, after administering the test substance to the mammal, the blood glucose level of the mammal is measured, and the measured value is used to measure the blood glucose level in the mammal administered with the present protein such as the protein represented by SEQ ID NO: 2 instead of the test substance. By comparing the values, it can also be determined whether or not the test substance exhibits a hypoglycemic effect equivalent to or higher than that of the present protein such as the protein represented by SEQ ID NO: 2.
Further, after administering the test substance to the mammal, the blood glucose level of the mammal is measured, and the measured value is overexpressed in the mammal such as the protein represented by SEQ ID NO: 2 instead of the test substance. It is also possible to determine whether or not the test substance exhibits an insulin-like activity equal to or higher than that of the present protein such as the protein represented by SEQ ID NO: 2 by comparing the blood glucose levels. Methods for overexpressing the present protein in mammals include, for example, methods such as transgenic and adenovirus infection, and any method is known in the literature.

As described above, the test substance has 1) the ability of glucose uptake into muscle cells, 2) the ability to inhibit gluconeogenesis in hepatocytes, 3) the ability to activate insulin signals in muscle cells or hepatocytes, and 4) the administration to mammals. Insulin-like activity such as blood glucose-lowering ability can be assayed. Based on the ability evaluated by the assay method of the present invention, an insulin-like activity promoter, specifically, 1) a sugar uptake promoter into muscle cells, 2) a gluconeogenesis inhibitor in hepatocytes, 3) muscle cells or liver Insulin signal transduction promoters in cells and 4) hypoglycemic agents can be selected as sugar metabolism function improving agents, that is, therapeutic or preventive agents for sugar metabolism-related diseases (the search method of the present invention).
Specifically, when the insulin-like activity of the test substance is 1.2 times or more, preferably 1.5 times or more, more preferably 2 times or more, compared to the insulin-like activity of the present protein, It can be selected as a candidate substance for a therapeutic or prophylactic agent for a sugar metabolism-related disease having an activity for improving the sugar metabolism function, specifically, an insulin signal transduction promoting activity.
For example, the sugar uptake promotion rate indicating the ability of the test substance to take up sugar uptake is a substance showing a statistically significant value, specifically 1.2 times the sugar uptake uptake rate of the protein represented by SEQ ID NO: 2. Substances having the above values, more preferably 1.5 times or more, are selected as substances having the ability to promote sugar uptake.

The third aspect of the present invention is an assay for the insulin-like activity of a test substance using as an index whether the test substance promotes (induces) the expression of the present protein or a gene encoding the present protein (the present gene). Regarding the method. This will be described in detail below.
1) Assay method using the expression level of this gene as an index The present invention provides an assay method for insulin-like activity, which comprises the following steps (1) to (3). That is,
(1) a first step of contacting a test substance with a cell capable of expressing a gene encoding this protein;
(2) a second step of measuring an expression level of the gene in a cell contacted with a test substance and comparing the expression level with an expression level of the gene in a control cell not contacted with the test substance; and (3) A third step of evaluating the insulin-like activity of the test substance using as an index whether or not the test substance increases the expression level of the gene based on the comparison result of the second process.
Here, the same thing as the above is mentioned as a test substance.
The “cell capable of expressing the gene encoding this protein (this gene)” is not particularly limited as long as it is a cell that expresses this gene, and a cell capable of expressing this gene may be used as it is. You may use the cell transformed with the vector containing a gene. Examples of the derived animal species include rodent mammals such as rats, mice and guinea pigs, dogs, monkeys and humans.
Specifically, HEK293, which is a human kidney-derived cell, CHO-K1, which is a hamster ovary-derived cell, or COS-1 or COS-7, which is an African green monkey kidney-derived cell. In addition, mammalian fat-derived cells, liver-derived cells, muscle-derived cells, and the like may be used, and examples thereof include 3T3-L1, which is an established adipocyte, and HepG2, which is an established hepatocyte.

  Detection and quantification of the expression level of this gene can be performed by a known method such as Northern blotting or RT-PCR using RNA prepared from the cells or a complementary polynucleotide transcribed therefrom. Specifically, by using a polynucleotide having at least 15 consecutive nucleotides in the nucleotide sequence of the protein gene and / or its complementary polynucleotide as a primer or probe, the presence or absence of expression of the gene in RNA and its The expression level can be detected and measured. Such a probe or primer is based on, for example, primer 3 (HYPERLINK http://www.genome.wi.mit.edu/cgi-bin/primer/primer3.cgi http: / /www.genome.wi.mit.edu/cgi-bin/primer/primer3.cgi) or vector NTI (manufactured by Infomax).

When using Northern blotting, the primers or probes radioisotope (such as ³² P, ³³ P: RI) or a fluorescent substance labeled with a, it, RNA transfer cells derived from a nylon membrane or the like according to a conventional method After hybridization, the formed duplex of the primer or probe (DNA or RNA) and RNA is used as a signal from a label (RI or fluorescent substance) of the primer or probe as a radiation detector ( A method of detecting and measuring with a BAS-1800II (manufactured by Fuji Film) or a fluorescence detector can be exemplified. Further, using the AlkPhos Direct Labeling and Detection System (manufactured by Amersham Pharamcia Biotech), the probe is labeled in accordance with the protocol, hybridized with cell-derived RNA, and then the signal derived from the labeled product of the probe is multibiotic. A method of detecting and measuring with Major STORM860 (manufactured by Amersham Pharmacia Biotech) can also be used.

  When using the RT-PCR method, cDNA was prepared from cell-derived RNA according to a conventional method, and was prepared based on the sequence of this protein gene so that the target protein gene region could be amplified using this as a template. A pair of primers (a normal strand that binds to the cDNA (− strand) and a reverse strand that binds to the + strand) are hybridized with this, and PCR is performed according to a conventional method, and the resulting amplified double-stranded DNA is detected. The method of doing can be illustrated. In addition, the detection of the amplified double-stranded DNA was performed by a method for detecting the labeled double-stranded DNA produced by performing the PCR using a primer previously labeled with RI or a fluorescent substance. For example, a method may be used in which double-stranded DNA is transferred to a nylon membrane or the like according to a conventional method, and the labeled primer is used as a probe to hybridize with this to detect it. The produced labeled double-stranded DNA product can be measured with an Agilent 2100 Bioanalyzer (manufactured by Yokogawa Analytical Systems). In addition, prepare an RT-PCR reaction solution according to the protocol using SYBR Green RT-PCR Reagents (Applied Biosystems) and react with ABI PRIME 7900 Sequence Detection System (Applied Biosystems) to detect the reaction product. You can also.

2) Assay method using the expression level of the present protein as an indicator The present invention provides an assay method for insulin-like activity, comprising the following steps (1) to (3). Ie:
(1) a first step of contacting a test substance with a cell capable of expressing the protein;
(2) a second step of measuring the expression level of the protein in a cell contacted with a test substance, and comparing the expression level with the expression level of the protein in a control cell not contacted with the test substance; A third step of evaluating the insulin-like activity of the test substance using as an index whether or not the test substance increases the expression level of the protein based on the comparison result of the second step.
Here, examples of the test substance include the same substances as described above. Examples of the “cell capable of expressing the present protein” include the same cells as the “cell capable of expressing the gene encoding the present protein (present gene)” in 1) above.
The expression level of the protein can be detected and quantified according to a known method such as Western blotting using an antibody that recognizes the protein. Western blotting uses an antibody that recognizes this protein as a primary antibody, followed by a secondary antibody labeled with a radioactive isotope such as ¹²⁵ I, a fluorescent substance, an enzyme such as horseradish peroxidase (HRP), etc. It can be carried out by labeling with a binding antibody and measuring a signal derived from these labeling substances with a radiation measuring instrument (BAI-1800II: manufactured by Fuji Film Co., Ltd.), a fluorescence detector or the like. Moreover, after using the antibody which recognizes this protein as a primary antibody, it detects according to the said protocol using ECL Plus Western Blotting Detection System (made by Amersham Pharmacia Biotech), and multi-bio major STORM860 (made by Amersham Pharmacia Biotech) Can also be measured.

  The form of the antibody is not particularly limited, and may be a polyclonal antibody using the present protein as an immunizing antigen or a monoclonal antibody thereof, and further, at least a continuous amino acid sequence constituting the present protein. An antibody having antigen-binding property to a polypeptide usually consisting of 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids is also included in the antibody of the present invention.

  Methods for producing these antibodies are already well known, and the antibodies of the present invention can also be produced according to these conventional methods (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.12-11.13).

3) Assay method using a reporter gene assay using the expression control region of the present gene The present invention provides an assay method for insulin-like activity, comprising the following steps (1) to (3). Ie:
(1) a first step of bringing a test substance into contact with a cell containing a reporter gene operably linked to an expression control region of a gene encoding this protein;
(2) a second step of measuring the expression level of the reporter gene in a cell contacted with a test substance, and comparing the expression level with the expression level of the gene in a control cell not contacted with the test substance; and (3) A third step of evaluating the insulin-like activity of the test substance using as an index whether or not to increase the expression level of the reporter gene based on the comparison result of the second step.
Here, the test substance includes the same substances as described above.
“The expression control region of a gene encoding this protein (this gene)” usually refers to a range from several kb to several tens of kb upstream of the chromosomal gene, for example, (i) 5′-race method (5 ′ -RACE method) (for example, 5'full Race Core Kit (manufactured by Takara Shuzo Co., Ltd.) etc.), a step of determining the 5 'end by a usual method such as oligo cap method, S1 primer mapping; ii) A 5′-upstream region is obtained using a Genome Walker Kit (manufactured by Clontech) or the like, and the obtained upstream region can be identified by a technique including a step of measuring promoter activity.

  A reporter gene formed by linking the expression control region of the protein gene in a functional manner may be prepared by a method known to those skilled in the art. That is, the conventional genetic engineering described in `` Molecular Cloning: A Laboratory Manual 2nd edition '' (1989), Cold Spring Harbor Laboratory Press, `` Current Protocols In Molecular Biology '' (1987), John Wiley & Sons, Inc. The expression control region of this protein gene excised according to the technique can be incorporated on a plasmid containing a reporter gene.

Examples of reporter genes include glucuronidase (GUS), luciferase, chloramphenicol transacetylase (CAT), β-galactosidase, and green fluorescent protein (GFP).
A reporter gene that is operably linked to the prepared expression control region of the protein gene is inserted into a vector that can be used in a cell into which the reporter gene is to be introduced, using a normal genetic engineering technique, and a plasmid Can be prepared and introduced into a suitable host cell. Transformed cells can be obtained by culturing in a medium under selection conditions according to the reporter gene.

  In addition, as a method for measuring the expression level of the reporter gene, a method corresponding to each reporter gene may be used. For example, when a luciferase gene is used as a reporter gene, the transformed cell is cultured for several days, an extract of the cell is obtained, and then the extract is reacted with luciferin and ATP to cause chemiluminescence, and the luminescence intensity Promoter activity can be detected by measuring. In this case, a commercially available luciferase reaction detection kit such as Picker Gene Dual Kit (registered trademark; manufactured by Toyo Ink) can be used.

  In the above 1) to 3), a “cell capable of expressing a gene encoding the present protein”, “a cell capable of expressing the present protein” or “an expression control region of the present gene linked in a functional manner” The “cell containing the gene” and the test substance may be contacted while culturing under conditions that allow the cell to grow. For example, in the case of the transformed cell of the present invention using a mammalian cell as a host, a fetal bovine is appropriately used. It can be cultured in a commercially available medium such as D-MEM, OPTI-MEM, RPMI1640 medium (Gibco-BRL) supplemented with serum derived from mammals such as serum.

  In the above 1) to 3), “control cells not to be contacted with a test substance” means “cells capable of expressing a gene encoding the present protein” and “cells capable of expressing the present protein” used in each first step. Alternatively, “a cell containing a reporter gene operably linked to the expression control region of this gene” refers to the cell when the test substance is not contacted. “When the test substance is not contacted”, the same amount of solvent (blank) as the test substance is added instead of the test substance, or the negative control substance that does not affect the expression of the gene, protein or reporter gene Is also included.

In the above 1) to 3), a test substance having an activity of inducing expression of the gene or the protein is selected based on the expression level of the gene, the protein or the reporter gene measured in the first step and the second step. Can do. That is, the expression level of the present gene, the present protein or the reporter gene in the cells to which the test substance is added is 1.2 times or more, preferably 1.5 times or more, compared to the expression level in the control cells to which no test substance is added. If it is preferably 2 times or more, the test substance can be selected as an expression promoter (expression inducer) of this gene or protein (search method of the present invention).
The expression inducer of the present gene or the present protein selected as described above also has an activity to improve the glucose metabolism function, specifically, an insulin signal transduction promoting activity of the therapeutic or preventive agent for a sugar metabolism-related disease of the present invention. Candidate substance.

  The present protein and fragments thereof, or compounds found by the search method of the present invention are used as they are or as known pharmaceutical acceptable carriers (excipients, diluents, extenders, binders, lubricants). And a pharmaceutical composition by mixing with conventional additives and the like. The pharmaceutical composition is prepared in an orally administered form such as tablets, pills, capsules, powders, granules, syrups, emulsions and suspensions; injections, drops, external preparations, suppositories, etc. Depending on the parenteral administration agent, etc., it can be systemically or locally administered orally or parenterally. In the case of parenteral administration, intravenous administration, intradermal administration, subcutaneous administration, rectal administration, transdermal administration, and the like are possible.

The appropriate dosage form is an active ingredient (this protein, or a substance selected by the search method of the present invention or a pharmaceutically acceptable substance thereof) in an acceptable normal carrier, excipient, binder, stabilizer, diluent and the like. It can be produced by blending acceptable salts and the like. When used in an injection form, acceptable buffering agents, solubilizing agents, isotonic agents and the like can be added.
The dosage varies depending on the type of active ingredient, the route of administration, the age, weight, and symptoms of the subject or patient, but it cannot be generally defined. About 1 mg to 2 g, preferably about 5 mg to several tens mg can be administered in one to several times a day. In the case of injection, it is sufficient to administer about 0.1 mg to about 500 mg as an active ingredient amount in an adult, and the daily dose can be administered once or divided into several times.

  Examples of the active ingredient substance include the gene itself. In this case, gene therapy may be performed by incorporating the gene into a gene therapy vector. Also in these cases, the dosage and administration method of the gene therapy composition vary depending on the patient's weight, age, symptoms, etc., and can be appropriately selected by those skilled in the art.

  When the gene therapy is described in detail, the gene therapy is carried out in the same manner as in this type of gene therapy, for example, a mammal (patient) directly suffering from a disease related to glucose metabolism, for example, the gene or a chemical modification thereof. Can be carried out by a method of controlling the expression of the target gene by administering it into the body of the patient, or a method of controlling the expression of the target gene by the cell by introducing these genes into the target cells of the patient.

Here, as the chemical modification, for example, phosphorothioate, phosphorodithioate, alkyl phosphotriester, alkyl phosphonate, alkyl phosphoamidate, etc., derivatives that can enhance the ability to migrate into the cell or the stability in the cell. ("Antisense RNA and DNA" published by WILEY-LISS, 1992, pp. 1-50, J. Med. Chem. 36: 1923-1937, 1993). These can be synthesized according to conventional methods.
The gene can be formulated using a stabilizer, buffer, solvent, or the like that is commonly used for administration.

  In the method of introducing the present gene into a target cell of a patient, the polynucleotide used is preferably 100 bases or more, more preferably 300 bases or more, and even more preferably 500 bases or more. In addition, this method includes an in vivo method for introducing a gene into cells in a living body and an ex vivo method for introducing a gene into a cell once taken out of the body and returning the cell to the body (Nikkei Science, 1994 4). Monthly issue, pages 20-45, monthly pharmacy, 36 (1), 23-48 (1994), experimental medicine special edition, 12 (15), all pages (1994)). Among these, the in vivo method is preferable, and there are a viral introduction method (method using a recombinant virus) and a non-viral introduction method (see the above-mentioned documents).

  As a method of using the above-mentioned recombinant virus, for example, a polynucleotide of this gene is incorporated into a viral genome such as a retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poliovirus, symbis virus, etc. The method to introduce is mentioned. Of these, methods using retroviruses, adenoviruses, adeno-associated viruses and the like are particularly preferred. Examples of non-viral introduction methods include the liposome method and the lipofectin method, and the liposome method is particularly preferable. Other non-viral introduction methods include, for example, a microinjection method, a calcium phosphate method, an electroporation method, and the like.

The pharmaceutical composition for gene therapy comprises the present gene or a recombinant virus containing these genes and infected cells into which these viruses have been introduced as active ingredients. The administration form, administration route and the like of the composition to a patient can be appropriately determined according to the disease or symptom to be treated. For example, it can be administered in an appropriate dosage form such as an injection, intravenously, arterial, subcutaneously, intramuscularly, etc., or can be directly administered and introduced into a disease target site of a patient. In the case of employing the in vivo method, the gene therapy composition includes, for example, a form in which a viral vector containing the gene is embedded in liposomes or membrane fusion liposomes in addition to the administration form such as an injection containing the gene (Sendai). Virus (HVJ) -liposomes). These liposome preparation forms include suspensions, freezing agents, centrifugal concentrated freezing agents, and the like. Moreover, the composition for gene therapy can also be made into the form of the cell culture solution infected with the virus which introduce | transduced the vector containing this gene. The dosage of the active ingredient in these various forms of preparation can be appropriately adjusted depending on the degree of the disease to be treated, the age of the patient, the body weight, and the like. In general, about 0.0001-100 mg, preferably about 0.001-10 mg per adult patient may be administered once every several days to several months. In the case of a retroviral vector containing this gene, the retroviral titer can be selected from an amount range of about 1 × 10 ³ pfu-1 × 10 ¹⁵ pfu per kg patient body weight per day. In the case of cells into which this gene has been introduced, about 1 × 10 ⁴ cells / body-1 × 10 ¹⁵ cells / body may be administered.

  EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

(Isolation of this gene: isolation of a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2)
Using 1.0 μg of total RNA prepared from human abdominal adipose tissue by the guanidine thiocyanate / cesium chloride method (Chirgwin, JM et al., Biochemistry, 18, 5294, 1979) as a template, and a cDNA synthesis kit (Takara Shuzo) ) And the oligo dT primer attached thereto, 50 units of MMTV reverse transcriptase (Takara Shuzo) were added in the presence of 1 mM dNTP, and at room temperature for 10 minutes, then 42 ° C., 15 minutes, further 99 ° C., 5 Single-stranded cDNA was synthesized by incubating for minutes.
Subsequently, using 2.0 μg of the single-stranded cDNA as a template, 20 μmol each of the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 3 and the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 4 as a primer, 200 μM dNTP, 1 unit of DNA polymerase (manufactured by PerkinElmer) was added in the presence of 1.5 mM MgCl ₂ , and the PCR reaction was carried out under conditions of 55 cycles at 94 ° C. for 1 minute, further 72 ° C. for 2 minutes. went. The obtained PCR reaction product was subjected to 1% agarose gel electrophoresis (electrophoresis buffer; Tris-borate buffer (manufactured by Nacalai Tesque)), and a DNA band of about 1.5 kbp was excised from the gel, and Sambrook, J., Fritsch, EF, Maniatis, T .: “Molecular Cloning Second Edition”, cloned into the HincII site of plasmid vector pUC118 (Takara Shuzo) by the method described in Cold Spring Harbor Laboratory Press (1989). The base sequence of the cloned DNA was determined with a 373A type DNA sequencer manufactured by Applied Biosystems using Taq Dye Primer Cycle Sequencing Kit and Taq Dye Deoxy Terminator Cycle Sequencing Kit (Applied Biosystems). The DNA consisted of the base sequence represented by SEQ ID NO: 1, and the base sequence encoded the amino acid sequence represented by SEQ ID NO: 2.
Thus, a gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 was isolated.

(Preparation of protein expression plasmid consisting of amino acid sequence shown in SEQ ID NO: 2)
An expression plasmid for producing a protein having an amino acid sequence represented by amino acids from the first amino acid to the 491st amino acid in the amino acid sequence represented by SEQ ID NO: 2 is the DNA amplified and isolated in Example 1. A known expression vector (pcDL-SRα296: Takebe, Y., Seiki, M., Fujisawa, J., Hoy, P., Yokota, K., Arai, K., Yoshida, M , and Arai, N., Mol. Cell Biol. Jan. 1988, p466-472). With the expression plasmid thus prepared, monkey-derived COS1 cells (manufactured by ATCC) were transformed using the Fugen reagent (manufactured by Boehringer) in the same manner as described in the protocol attached to the reagent.

Example 3 (Preparation of the present protein: Preparation of a protein preparation comprising the amino acid sequence represented by SEQ ID NO: 2)
A protein having an amino acid sequence represented by amino acids from the first amino acid to the 491st amino acid in the amino acid sequence represented by SEQ ID NO: 2 is obtained by treating monkey-derived COS1 cells transformed in Example 2 with penicillin, streptomycin ( GIBCO) was secreted into OPTI-MEM medium (GIBCO) to which final concentrations of 100 units / ml and 100 μg / ml were added, respectively, to secrete the protein into the medium, and on the second day after transformation To cell culture supernatants from day 8 to day 8 were collected. The culture supernatant was filtered through an ultrafiltration membrane, and purified sequentially by DEAEsepharose, ANX-Sepharose, Octyl-Sepharose, and Mono-Q columns (all manufactured by Amersham Pharmacia). The finally obtained protein preparation was a single band in terms of silver staining.

(Test on sugar uptake of this protein in muscle cells)
(1) Culture of muscle cells Dulbecco supplemented with sleeping L6 cells with FBS (GIBCO), penicillin and streptomycin (GIBCO) to final concentrations of 10%, 100 units / ml and 100 μg / ml, respectively. The suspension was suspended in 30 ml of a modified Eagle medium (containing 4.5 g / L D-glucose and 584 mg / L L-glutamine; manufactured by GIBCO) (hereinafter referred to as FBS-containing medium). It was injected into T150 for cells (manufactured by Iwaki Glass) and cultured overnight at 37 ° C. in the presence of 5% CO ₂ . The cells were treated with 15 mL of phosphate buffer (0.20 g / L KCl, 0.20 g / L KH ₂ PO ₄ , 8.00 g / L NaCl, 2.16 g / L Na ₂ HPO ₄ · 7H ₂ O, hereinafter PBS In the flask, trypsin-ethylenediaminetetraacetic acid tetrasodium (hereinafter referred to as EDTA · 4Na) solution (containing 0.05% trypsin, 0.53 mM EDTA · 4Na, manufactured by GIBCO) 1 to 2 ml. Was added so that the cells were immersed, and left at 37 ° C. for 5 minutes. To this, an FBS-containing medium was added in an amount about 10 times that of a trypsin-EDTA · 4Na solution to obtain a cell suspension. The cell suspension was poured into a 12-well plate at a concentration of 1 × 10 ⁵ cells / well and cultured at 37 ° C. in the presence of 5% CO ₂ . Two days later, the medium was replaced with an FBS-containing medium containing 2% FBS, and further cultured for 5 days.
(2) Measurement of sugar uptake activity The well containing the test cell obtained in (1) was washed with a phosphate buffer, and only FBS was removed from the FBS-containing medium (hereinafter referred to as serum-free medium). And cultured for 18 hours. KRB buffer well (25mM Hepes (pH7.4), 120mM NaCl, 5mM KCl, 1.2mM MgSO4,1.3mM CaCl 2, 1.2mM K2HPO4,23.8mM sodium hydrogen carbonate, sodium 0.1mM pyruvate, Washed with 0.1% BSA, 0.22 μm filter), added 500 μL of KRB buffer, and incubated at 37 ° C. for 30 minutes. The buffer solution in the wells was removed, the preparation of the present protein obtained in Example 3 was added to 500 μL of KRB buffer so as to be 0 to 30 nM, and further incubated at 37 ° C. for 30 minutes. Furthermore, 10 μL of 2-deoxy-D- [1- ³ H] glucose (manufactured by Amersham) diluted to 0.01 μCi / μL with KRB buffer was added and incubated at 37 ° C. for 10 minutes. The solution in the well was removed, and 1 mL of phosphate buffer that had been ice-cooled in advance was added to wash the well. Further, the well was washed once with a phosphate buffer, and then 300 μL of 1N NaOH was added and stirred well. 250 μL of the liquid was collected from the well, added to a vial bottle into which 4 ml of a liquid scintillator (manufactured by Packard) was previously injected, and the total radioactivity was measured with a liquid scintillation counter (manufactured by Packard) (hereinafter referred to as measured value 1). .) The same procedure was performed except that a phosphate buffer was added instead of the present protein sample to the well containing the test cell obtained in Example 5 (1), and the total radioactivity was measured (hereinafter, measured value). From these measured values, the sugar uptake promotion rate of this protein was calculated according to the following formula.
Sugar uptake promotion rate (%) = (measured value 1 / measured value 2) × 100
The rate of acceleration of sugar uptake by 3 nM, 10 nM and 30 nM of this protein preparation was 116%, 139% and 145%, respectively.
Moreover, as a result of calculating the sugar uptake promotion rate by insulin according to the same method as described above except that 3 nM, 10 nM and 30 nM insulin was added instead of the protein preparation, 100%, 138% and 150%.

(Measurement of insulin signal activation rate in muscle cells of this protein)
The L6 cells that had fallen asleep in Example 4 (1) were seeded in a 90 cm dish and cultured in an FBS-containing medium until confluent. After removing the medium, the medium was washed with a phosphate buffer, cultured in a serum-free medium for 20 hours, and then the medium was removed again. Incubation was carried out at 37 ° C. for 10 minutes in a serum-free medium containing 100 ng / mL of the present protein preparation obtained in Example 3. The reaction was stopped by removing the medium, and the cells were washed twice with an ice-cold phosphate buffer. After freezing the cells with liquid nitrogen, 1 mL of cell disruption buffer (20 mM Tris-HCl (pH 7.4), 2 mM CaCl 2, 1 mM MgCl 2, 140 mM NaCl, 1% NP-40, 2 μg / mL aprotinin, 10 μg / mL The cells were disrupted by adding leupeptin, 1 mM PMSF and 2 mM Na ₃ VO ₄ ), and then centrifuged at 15,000 g for 30 minutes at 4 ° C. to obtain a cell disruption supernatant. Anti-IRS-1 antibody # 06-248 (Upstate Biotechnology) 5 μg was previously bound to Protein G-sepharose (Amersham Pharmacia) 20 μL, mixed with 1 mL of the resulting cell disruption supernatant, and incubated at 4 ° C. overnight. did. On the next day, the beads were washed several times with a cell disruption buffer, and a sample buffer for SDS-PAGE (60 mM Tris-HCl (pH 6.7), 3% SDS, 2% mercaptoethanol, 5% glycerol) was added and 100 was added. Proteins bound to the beads were eluted by heating at 5 ° C. for 5 minutes. The eluted protein was separated by SDS-PAGE (Daiichi Kagaku) and immunoblotted using anti-phosphotyrosine antibody RC20 (BD Bioscience) or anti-PI3 kinase p85 antibody # 06-195 (Upstate Biotechnology). Then, phosphorylated IRS-1 and phosphorylated PI3 kinase protein were respectively detected and quantified using an ECL detection kit (manufactured by Amersham).
In addition, 10 μL of cell disruption supernatant was directly separated by SDS-PAGE (Daiichi Kagaku), immunoblotted using anti-phosphorylated Akt antibody # 9271 (Cell Signaling), and ECL detection kit (Amersham) ) To detect and quantify the phosphorylated Akt protein.
As a result, when this protein preparation was added, phosphorylation of IRS-1, PI3 kinase, and Akt molecule was promoted as compared to the negative control without addition (FIG. 1). As a positive control, each phosphorylated protein signal in the case of adding 100 ng / mL insulin instead of this protein is also shown.

(Test on inhibition of gluconeogenesis in hepatocytes by this protein)
(1) Preparation of test cells H4IIE cells (purchased from Dainippon Pharmaceutical Co., Ltd.) were mixed with FBS, penicillin, streptomycin, cAMP (Sigma), and dexamethasone (Sigma) at a final concentration of 10%, 100 units / ml, 100 μg / The cells were cultured in Dulbecco's modified Eagle medium (containing 4.5 g / L glucose and 584 mg / L L-glutamine, manufactured by GIBCO) (hereinafter referred to as a medium for hepatocytes) added to a concentration of ml, 50 μM and 100 nM. Cells were collected from the incubator by the method described in Example 4, seeded in 12-well plates at a concentration of 3 × 10 ⁵ cells / well, and cultured at 37 ° C. in the presence of 5% CO ₂ . The culture was continued until the cells became confluent. The cells were washed with a phosphate buffer, replaced with a medium for hepatocytes containing 5 mM glucose, and cultured for 24 hours.
The cells were washed twice with a phosphate buffer, and 1 mL of gluconeogenesis buffer solution (8.3 g / L DMEM-base, 4 mM L-glutamine, 1 mM pyruvin) containing 0 to 1 nM of the present protein sample obtained in Example 3 was used. Cell cultures overnight, replacing with sodium acid, 3.7 g / L sodium bicarbonate, 0.9 g / L lactic acid, 100 units / mL penicillin, and 100 μg / mL streptomycin, filtered through a 0.22 μm filter). The next day, the cell supernatant was collected. The sugar (glucose) concentration contained in the culture supernatant collected according to the protocol was measured using F-kit glucose (JK International).
About the gluconeogenesis promotion test by the insulin which is a positive control, it measured by performing the same operation except adding 0-1 nM insulin instead of this protein sample obtained in Example 3.
As a result, the gluconeogenesis in each concentration of the protein preparation when the sugar concentration in the medium without addition of the protein preparation, that is, the amount of gluconeogenesis of 4.01 fmol / cell / hour is taken as 100%. The amounts were 98%, 32%, and 3% at 0.01 nM, 0.1 nM, and 1 nM, respectively, and it was confirmed that this protein inhibited gluconeogenesis of hepatocytes in a concentration-dependent manner. On the other hand, in 0.01 nM, 0.1 nM and 1 nM insulin, they were 82%, 20% and 4%, respectively.

(Measurement of insulin action promotion rate in hepatocytes of this protein)
H4IIE cells (manufactured by Dainippon Pharmaceutical Co., Ltd.) were cultured in a hepatocyte culture medium in the same manner as in Example 6, spread in a 90 mm dish, and cultured until the cells were confluent. After removing the medium, the medium was washed with a phosphate buffer, cultured in a hepatocyte-free medium (hereinafter, serum-free hepatocyte medium) containing no FBS for 20 hours, and then the medium was removed again. Further, it was incubated at 37 ° C. for 10 minutes in a serum-free hepatocyte medium containing 100 ng / mL of the present protein preparation. The reaction was stopped by removing the medium, and the cells were washed twice with an ice-cold phosphate buffer. Subsequent operations were performed by the method described in Example 5 to detect and quantify each phosphorylated molecule.
As a result, when the present protein preparation obtained in Example 3 was added, phosphorylation of IRS-1, PI3 kinase, and Akt molecules was promoted as compared to the negative control without addition (FIG. 2). . As a positive control, each phosphorylated protein signal in the case of adding 100 ng / mL insulin instead of this protein is also shown.

(Test 1 on the regulation of glucose metabolism in vivo by this protein)
Under anesthesia of 10-week-old male KKAy mice (Claire Japan), phosphate buffer (negative control), 487 pmol of this protein preparation, or 487 pmol of insulin was administered from the tail vein. Blood was collected from the orbit of the mice before administration and 15 minutes, 30 minutes, 45 minutes, 60 minutes and 120 minutes after administration. The sugar concentration in the collected blood was measured using F-kit glucose (JK International) according to the protocol. Table 1 shows the blood glucose level (average value of each group n = 4) of KKAy mice.
As a result of the above, as shown in Table 1 and FIG. 3, the present protein exhibited a hypoglycemic effect equivalent to that of insulin used as a positive control.

(Test 2 on the regulation of glucose metabolism in vivo by this protein)
(1) Production of recombinant adenovirus The DNA amplified and isolated in Example 1, that is, the recombinant virus particle having the gene encoding this protein, was prepared in the kit using an adenovirus expression vector kit (Takara Shuzo). Prepared according to the method described. As a negative control having no amplified or isolated DNA in Example 1, β-galactosidase gene-containing virus particles attached to the kit were used. HEK293 cells (American Type Culture Collection) were infected with virus particles and amplified three times, and a layer of virus solution obtained by CsCl density ultracentrifugation at 100,000 g × 90 minutes was extracted with a syringe. The obtained partially purified virus solution was diluted with 10 mM Tris-HCl, 1 mM EDTA (pH 8.0), and subjected to CsCl density gradient ultracentrifugation (100,000 g × 16 hours), and the virus solution layer was again collected. The obtained purified virus solution was dialyzed against 10 mM Tris-HCl (pH 8.0), 2 mM MgCl 2, 5% glucose, and stored frozen at −80 ° C.

(2) In vivo glucose metabolism control test of this protein by adenovirus administration
8-week-old male C57BL / 6J mice and 10-week-old male KKAy mice (Claire Japan) were inoculated with 1.0 × 10 ⁸ units of the virus produced in (1) per mouse, and then the breeding was continued. Blood was collected from the mice after one day. The sugar concentration in the blood was measured according to the protocol using F-kit glucose (JK International). The test was conducted with 5 animals in each group.
As a result, as shown in Table 2 and FIG. 4, when a virus having this gene was infected, no significant blood glucose level reduction occurred in C57BL / 6J mice, whereas genetic diabetes was observed. An effect of lowering blood glucose level in KKAy mice that developed and was hyperglycemic was observed (p <0.007).

It is a figure which shows phosphorylation of IRS-1, PI3 kinase, and an Akt molecule | numerator in a muscle cell. It is a figure which shows phosphorylation of IRS-1, PI3 kinase, and an Akt molecule | numerator in a hepatocyte. KKAy mice were administered with the present protein preparation or insulin obtained in Example 3, and blood glucose levels (n = 4 for each group) before administration and 15 minutes, 30 minutes, 45 minutes, 60 minutes and 120 minutes after administration. It is a figure which shows the result of having measured (average value). It is a figure which shows the result of having infected a KKAy mouse | mouth with the virus which has this protein gene obtained in Example 9, and measuring a blood glucose level.

SEQ ID NO: 3
Oligonucleotide primer designed to amplify the gene SEQ ID NO: 4
Oligonucleotide primers designed to amplify genes

Claims (24)

A therapeutic or prophylactic agent for a sugar metabolism-related disease, comprising as an active ingredient a protein consisting of any of the following amino acid sequences (a) to (h) or a fragment thereof;
<Amino acid sequence group>
(A) the amino acid sequence represented by SEQ ID NO: 2,
(B) an amino acid sequence in which one or more amino acids are deleted, added or substituted in the amino acid sequence represented by SEQ ID NO: 2;
(C) a partial amino acid sequence obtained by deleting 26 amino acids from the amino terminus in the amino acid sequence represented by SEQ ID NO: 2;
(D) an amino acid sequence in which methionine is added to the amino terminus of the amino acid sequence of (c),
(E) an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2,
(F) an amino acid sequence encoded by DNA having a base sequence represented by nucleotides from the 18th nucleotide to the 1493th nucleotide in the base sequence represented by SEQ ID NO: 1;
(G) It is encoded by DNA having a base sequence having 80% or more sequence identity with DNA having the base sequence shown by nucleotides 18 to 1493 in the base sequence shown by SEQ ID NO: 1. And an amino acid sequence having insulin-like activity,
(H) Hybridizes under stringent conditions with DNA having complementarity to DNA having the nucleotide sequence represented by nucleotides from the 18th nucleotide to the 1493th nucleotide in the nucleotide sequence represented by SEQ ID NO: 1. An amino acid sequence encoded by DNA and having insulin-like activity. The therapeutic or prophylactic agent according to claim 1, which enhances insulin-like activity. The therapeutic or prophylactic agent according to claim 2, wherein the insulin-like activity is insulin signal transduction promoting activity in hepatocytes or muscle cells. The therapeutic or preventive agent according to claim 2, wherein the insulin-like activity is gluconeogenesis-inhibiting activity in hepatocytes or saccharide uptake promoting activity in muscle cells. The therapeutic or prophylactic agent according to claim 2, wherein the insulin-like activity is a hypoglycemic action. A method for assaying insulin-like activity,
(1) a first step of bringing a test substance into contact with muscle cells or hepatocytes,
(2) The second step of measuring insulin-like activity in the cell, and (3) the measurement value measured in the second step is any one of the following (a) to (h) instead of the test substance Compared with the measured value when the first step and the second step are performed using a protein consisting of an amino acid sequence, the indicator is that the insulin-like activity of the test substance is equal to or greater than the insulin-like activity of the protein, A third step of evaluating the insulin-like activity of the test substance,
A method for assaying insulin-like activity, comprising:
<Amino acid sequence group>
(A) the amino acid sequence represented by SEQ ID NO: 2,
(B) an amino acid sequence in which one or more amino acids are deleted, added or substituted in the amino acid sequence represented by SEQ ID NO: 2;
(C) a partial amino acid sequence obtained by deleting 26 amino acids from the amino terminus in the amino acid sequence represented by SEQ ID NO: 2;
(D) an amino acid sequence in which methionine is added to the amino terminus of the amino acid sequence of (c),
(E) an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2,
(F) an amino acid sequence encoded by DNA having a base sequence represented by nucleotides from the 18th nucleotide to the 1493th nucleotide in the base sequence represented by SEQ ID NO: 1;
(G) It is encoded by DNA having a base sequence having 80% or more sequence identity with the DNA having the base sequence shown by nucleotides 18 to 1493 in the base sequence shown by SEQ ID NO: 1. And an amino acid sequence having insulin-like activity,
(H) Hybridizes under stringent conditions with DNA having complementarity to DNA having the nucleotide sequence represented by nucleotides from the 18th nucleotide to the 1493th nucleotide in the nucleotide sequence represented by SEQ ID NO: 1. An amino acid sequence encoded by DNA and having insulin-like activity. The assay method according to claim 6, wherein the insulin-like activity is insulin signal transduction promoting activity. The assay method according to claim 7, wherein the insulin signal transduction promoting activity is measured using the amount of phosphorylated insulin receptor, IRS-1, PI3 kinase or Akt as an index. The assay method according to claim 6, wherein the insulin-like activity is gluconeogenesis-inhibiting activity in hepatocytes. The assay method according to claim 6, wherein the insulin-like activity is an activity of promoting sugar uptake into muscle cells. The assay method according to claim 6, wherein the muscle cell used in the first step is a skeletal muscle cell. The assay method according to any one of claims 6 to 9, wherein the hepatocytes used in the first step are established hepatocytes or primary cultured hepatocytes. Assay of insulin-like activity of a test substance using as an index whether the test substance promotes the expression of a protein consisting of any of the following amino acid sequences (a) to (h) or a gene encoding the protein: Method;
<Amino acid sequence group>
(A) the amino acid sequence represented by SEQ ID NO: 2,
(B) an amino acid sequence in which one or more amino acids are deleted, added or substituted in the amino acid sequence represented by SEQ ID NO: 2;
(C) a partial amino acid sequence obtained by deleting 26 amino acids from the amino terminus in the amino acid sequence represented by SEQ ID NO: 2;
(D) an amino acid sequence in which methionine is added to the amino terminus of the amino acid sequence of (c),
(E) an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2,
(F) an amino acid sequence encoded by DNA having a base sequence represented by nucleotides from the 18th nucleotide to the 1493th nucleotide in the base sequence represented by SEQ ID NO: 1;
(G) It is encoded by DNA having a base sequence having 80% or more sequence identity with DNA having the base sequence shown by nucleotides 18 to 1493 in the base sequence shown by SEQ ID NO: 1. And an amino acid sequence having insulin-like activity,
(H) Hybridizes under stringent conditions with DNA having complementarity to DNA having the nucleotide sequence represented by nucleotides from the 18th nucleotide to the 1493th nucleotide in the nucleotide sequence represented by SEQ ID NO: 1. An amino acid sequence encoded by DNA and having insulin-like activity. 14. The method for assaying insulin-like activity according to claim 13, comprising the following steps (1) to (3):
(1) a first step of bringing a test substance into contact with a cell capable of expressing a gene encoding the protein according to claim 13;
(2) a second step of measuring an expression level of the gene in a cell contacted with a test substance and comparing the expression level with an expression level of the gene in a control cell not contacted with the test substance; and (3) A third step of evaluating the insulin-like activity of the test substance using as an index whether or not the test substance enhances the expression of the gene based on the comparison result of the second step. 14. The method for assaying insulin-like activity according to claim 13, comprising the following steps (1) to (3):
(1) a first step of contacting a test substance with a cell capable of expressing the protein according to claim 13;
(2) a second step of measuring the expression level of the protein in a cell contacted with a test substance, and comparing the expression level with the expression level of the protein in a control cell not contacted with the test substance; A third step of evaluating the insulin-like activity of the test substance using as an index whether or not the test substance enhances the expression of the protein based on the comparison result of the second step. 14. The method for assaying insulin-like activity according to claim 13, comprising the following steps (1) to (3):
(1) a first step of bringing a test substance into contact with a cell containing a reporter gene operably linked to an expression control region of a gene encoding the protein according to claim 13;
(2) a second step of measuring the expression level of the reporter gene in a cell contacted with a test substance, and comparing the expression level with the expression level of the gene in a control cell not contacted with the test substance; and (3) A third step of evaluating the insulin-like activity of the test substance using as an index whether or not to increase the expression level of the reporter gene based on the comparison result of the second step. The assay method according to any one of claims 13 to 16, wherein the insulin-like activity is insulin signaling promoting activity. The assay method according to any one of claims 13 to 16, wherein the insulin-like activity is an activity of promoting sugar uptake into muscle cells. The assay method according to any one of claims 13 to 16, wherein the insulin-like activity is gluconeogenesis-inhibiting activity in hepatocytes. A candidate substance for a therapeutic or prophylactic agent for a glucose metabolism-related disease is selected using the insulin-like activity of the test substance as an index measured by the assay method according to any one of claims 6 to 19. A method for searching for a therapeutic or prophylactic agent for a sugar metabolism-related disease. 21. The search method according to claim 20, wherein the therapeutic or preventive agent for a glucose metabolism-related disease is an insulin signaling promoter in muscle cells or hepatocytes. The method for searching according to claim 20, wherein the therapeutic or preventive agent for a sugar metabolism-related disease is a sugar uptake promoter in muscle cells. The search method according to claim 20, wherein the therapeutic or preventive agent for a glucose metabolism-related disease is a gluconeogenesis inhibitor in the liver. A therapeutic or prophylactic agent for a sugar metabolism-related disease, comprising as an active ingredient a substance selected by the search method according to any one of claims 20 to 23.

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