JP4606766B2 - Therapeutic or preventive agent for diseases related to glucose metabolism - Google Patents

Description

  The present invention relates to a therapeutic or preventive agent for a sugar metabolism-related disease, and more particularly to a protein having a glucose tolerance improving activity or an insulin responsiveness promoting activity.

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, it is known that the protein consisting of the amino acid sequence represented by SEQ ID NO: 2 is a protein that is specifically expressed in the visceral adipose tissue of obese animals (see Non-Patent Document 1). However, the function of the protein in vivo is unknown, and the relationship between sugar metabolism-related diseases has not been known at all.
Hida K et al, J. Lipid Research, 41, p 1615-p1622 (2000)

  The problem to be solved by the present invention is to provide a therapeutic or preventive agent for a sugar metabolism-related disease, specifically, a protein having a glucose tolerance improving activity or an insulin responsiveness promoting activity.

  As a result of intensive studies under these circumstances, the present inventors have found that the protein comprising the amino acid sequence represented by SEQ ID NO: 2 has a glucose tolerance improving activity and an insulin responsiveness promoting activity, and is a therapeutic agent for a glucose metabolism-related disease. Or it discovered that it could become a preventive agent and came to complete this invention.

That is, the present invention
[1] A therapeutic or prophylactic agent for a sugar metabolism-related disease comprising a protein comprising any of the following amino acid sequences (hereinafter sometimes referred to as the present protein) or a fragment thereof as an active ingredient: :
<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, inserted or substituted in the amino acid sequence represented by SEQ ID NO: 2;
(C) an amino acid sequence represented by amino acids 22 to 414 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 224th nucleotide to the 1465th nucleotide in the base sequence represented by SEQ ID NO: 1,
(G) encoded by DNA having a base sequence having 80% or more sequence identity with the DNA having the base sequence shown by nucleotides 224 to 1465 in the base sequence shown by SEQ ID NO: 1 And an amino acid sequence having a glucose metabolism enhancing activity,
(H) Hybridizes under stringent conditions with DNA having complementarity to DNA having the nucleotide sequence represented by nucleotides from the first nucleotide to the 1242nd nucleotide in the nucleotide sequence represented by SEQ ID NO: 1. An amino acid sequence encoded by DNA and having an activity of promoting sugar metabolism;
[2] A therapeutic or preventive agent for a sugar metabolism-related disease, comprising as an active ingredient a gene encoding a protein consisting of an amino acid sequence selected from the amino acid sequence group described in [1];
[3] The therapeutic or preventive agent according to [1] or [2], which has glucose tolerance improving activity;
[4] The therapeutic or prophylactic agent according to [3], wherein the glucose tolerance improving activity is an insulin responsive promoting activity;
[5] Glucose tolerance of the test substance using as an index whether or not the test substance promotes the expression of a protein comprising an amino acid sequence selected from the amino acid sequence group described in [1] or a gene encoding the protein Assay method for improving activity;
[6] The method for assaying glucose tolerance improving activity according to [5], comprising the following steps (1) to (3):
(1) a first step of contacting a test substance with a cell capable of expressing a gene encoding a protein comprising an amino acid sequence selected from the amino acid sequence group described in [1],
(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 glucose tolerance improving 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 step;
[7] The method for assaying glucose tolerance improving activity according to [5], comprising the following steps (1) to (3):
(1) a first step of contacting a test substance with a cell capable of expressing a protein comprising an amino acid sequence selected from the amino acid sequence group described in [1],
(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 glucose tolerance improving 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;
[8] The method for assaying glucose tolerance improving activity according to [5], comprising the following steps (1) to (3):
(1) a test substance and a cell containing a reporter gene operatively linked to an expression control region of a gene encoding a protein comprising an amino acid sequence selected from the amino acid sequence group described in [1] The first step of contacting,
(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;
(3) A third step of evaluating the glucose tolerance improving 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;
[9] The assay method according to any one of [5] to [8], wherein the glucose tolerance improving activity is insulin responsiveness promoting activity;
[10] Selection of candidate substances for therapeutic or preventive agents for glucose metabolism-related diseases using as an index the glucose tolerance improving activity of the test substance measured by the assay method according to any one of [5] to [8] A method for searching for a therapeutic or prophylactic agent for a sugar metabolism-related disease,
[11] A therapeutic or prophylactic agent for a sugar metabolism-related disease, containing as an active ingredient a substance selected by the search method according to [10] or a pharmaceutically acceptable salt thereof;
[12] A therapeutic or preventive agent for a sugar metabolism-related disease, comprising as an active ingredient a substance that induces expression of a protein comprising an amino acid sequence selected from the amino acid sequence group described in [1], or a function promoting substance;
It is about.

  According to the present invention, a therapeutic or preventive agent for a glucose metabolism-related disease characterized by having a glucose tolerance improving activity or an insulin responsive promoting activity, a glucose tolerance improving activity test method, and a glucose metabolism related disease using the test method It has become possible to provide a method for searching for a therapeutic or preventive agent.

The present invention is described in detail below.
(I) Therapeutic or preventive agent The first aspect of the present invention relates to a therapeutic or prophylactic agent for a sugar metabolism-related disease containing the protein or fragment thereof as an active ingredient.
In the present specification, “a protein consisting of any of the following amino acid sequences” (the present protein) means (a) an amino acid sequence represented by SEQ ID NO: 2 and (b) an amino acid sequence represented by SEQ ID NO: 2 Or an amino acid sequence in which a plurality of amino acids are deleted, added, inserted or substituted, (c) an amino acid sequence represented by amino acids 22 to 414 in the amino acid sequence represented by SEQ ID NO: 2, (d) In the amino acid sequence of (c), an amino acid sequence in which methionine is added to the amino terminus, (e) an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, (f) SEQ ID NO: Having the nucleotide sequence represented by nucleotides 287 to 1465 in the nucleotide sequence represented by 1 (G) having 80% or more sequence identity with DNA having the nucleotide sequence represented by nucleotides 287 to 1465 in the nucleotide sequence represented by SEQ ID NO: 1 Complementary to an amino acid sequence encoded by a DNA having a base sequence, and (h) a DNA having a base sequence represented by nucleotides from the 287th nucleotide to the 1465th nucleotide in the base sequence represented by SEQ ID NO: 1. A protein having an amino acid sequence encoded by DNA hybridized under stringent conditions and having an activity of promoting sugar metabolism.

Here, “deletion, addition, insertion or substitution of amino acids” in (b) and “80% or more sequence identity” in (e) and (g) above are, for example, represented by SEQ ID NO: 2. This includes a naturally occurring mutation caused by processing of a protein having a specific amino acid sequence in a cell, 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 in which the protein's glucose metabolism enhancing activity 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. Such substitutions include, for example, i) glycine, alanine; ii) valine, isoleucine, leucine; iii) aspartic acid, glutamic acid, asparagine, glutamine, iv) serine, threonine; v) lysine, arginine; vi) phenylalanine, Substitution within the tyrosine group.

  The “amino acid sequence represented by the 22nd to 414th amino acids in the amino acid sequence represented by SEQ ID NO: 2” in the above (c) consists of the amino acid sequence represented by SEQ ID NO: 2 which is a secreted protein. In the protein, it corresponds to an amino acid sequence obtained by removing the partial amino acid sequence represented by the 21st amino acid from the 1st amino acid corresponding to the signal peptide portion.

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.

  Specific examples of the protein described above include a human protein consisting of the amino acid sequence shown in SEQ ID NO: 2, a rat homolog consisting of the amino acid sequence shown in SEQ ID NO: 4, and an amino acid sequence shown in SEQ ID NO: 6. A mouse type homolog can be mentioned.

  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 sugar metabolism enhancing activity of the protein. As long as it is not limited.

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 21 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 22 to 414 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 287th nucleotide to the 1465th nucleotide in the nucleotide sequence represented by SEQ ID NO: 1 A nucleotide sequence represented by nucleotides, and (VIII) a nucleotide having a sequence identity of 80% or more with a DNA having a nucleotide sequence represented by nucleotides 287 to 1465 in the nucleotide sequence represented by SEQ ID NO: 1. A DNA having a complementarity to a DNA having a nucleotide sequence represented by the nucleotide sequence from the 287th nucleotide to the 1465th 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 (eg, For example, according to Sambrook J., Frisch EF, Maniatis T., the method described in Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory press, etc.) To do. 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. The therapeutic agent or preventive agent for a sugar metabolism-related disease of the present invention is characterized by having glucose tolerance enhancing activity or insulin responsiveness promoting activity, that is, glucose tolerance improving activity.

As used herein, “glucose tolerance enhancing activity” is measured by carrying out OGTT (glucose tolerance test: a test for measuring changes in blood glucose level and insulin level after loading a certain amount of sugar in a mouse). This means that the decrease in blood glucose level after glucose load is enhanced in OGTT. Specifically, OGTT can be performed by the method described in Examples 9 and 10 of this specification.
In this specification, “insulin responsiveness” is measured by performing ITT (insulin tolerance test: measuring blood glucose level and insulin level fluctuation after loading a certain amount of insulin on a mouse). It is possible to express that the decrease in blood glucose level after insulin load is enhanced in ITT. Specifically, ITT can be performed by the method described in Examples 9 and 10 of the present specification.
In the OGTT and ITT, when the protein to be measured shows a blood glucose level comparable to that of the present protein used in Examples 9 and 10 of the present invention, the protein to be measured is “glucose tolerance enhancing activity” or “insulin response”. It can be said that it shows "sex".

(II) Assay method The second aspect of the present invention is the glucose tolerance improving activity of a test substance, which uses whether or not the test substance promotes the expression of the present protein or a gene encoding this protein (the present gene). It relates to the testing method.
(II-1) A method using the expression level of a gene encoding the protein as an index, ie, the following steps (1) to (3):
(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 the expression level of the 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) Based on the comparison result of the second step, the third step of evaluating the glucose tolerance improving activity of the test substance using as an index whether or not the test substance increases the expression level of the gene,
Have

The “gene encoding the present protein (hereinafter sometimes referred to as the present gene)” used in the first step is not particularly limited as long as it is a cell expressing the present gene. Either the sex gene or the gene introduced into the cell as a foreign gene may be used, but the endogenous gene of the cell to be used is preferred. When introduced as a foreign gene, this gene is preferably the gene of the animal species derived from the cell used.
Specific examples include adipose tissue-derived cells and transformed cells into which the present gene expression vector has been introduced. Examples of the derived animal species include rodent mammals such as rats, mice and guinea pigs, dogs, monkeys, humans and the like.

  Examples of the cells include cells isolated from animal tissues and organs, cells (tissues) forming a group having the same function / morphology, and the like, and may be cells in any differentiation process. .

  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 “control cell” in the second step refers to a cell that can express the present gene used in the first step and is not brought into contact with a test substance. “When the test substance is not contacted” includes the case where the same amount of solvent (blank) as the test substance is added instead of the test substance, or the case where a negative control substance that does not affect the expression of this gene is added. It is.

  In addition, the conditions for bringing the test substance into contact with the cells are not particularly limited, but it is preferable to employ conditions that do not greatly change the culture conditions (temperature, pH, medium composition, etc.) so that the cells do not die.

  The concentration of the test substance to be brought into contact with a cell capable of expressing the gene encoding this protein 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.

The “control cell not to be contacted with the test substance” refers to a cell that is not contacted with the test substance in the cell capable of expressing the gene used in the first step. “When the test substance is not contacted” includes the case where the same amount of solvent (blank) as the test substance is added instead of the test substance, or the case where a negative control substance that does not affect the expression of this gene is added. It is.
In addition, the conditions for bringing the test substance into contact with the cells are not particularly limited, but it is preferable to employ conditions that do not greatly change the culture conditions (temperature, pH, medium composition, etc.) so that the cells do not die.

  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 this gene and / or its complementary polynucleotide as a primer or probe, the presence or absence of expression of this gene in RNA or its expression The level can be detected and measured. Such a probe or primer is based on the base sequence of this gene, 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, a cDNA is prepared from cell-derived RNA according to a conventional method, and using this as a template, a target gene region can be amplified. A method in which a primer (a normal strand that binds to the cDNA (-strand), a reverse strand that binds to a + strand) is hybridized with this, and a PCR method is performed according to a conventional method, and the resulting amplified double-stranded DNA is detected. 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.

  If the expression of this gene in cells to which a test substance is added is 1.5 times or more, preferably 2 times or more, more preferably 3 times or more compared to the expression level in control cells to which no test substance is added, the test substance Can be selected as an expression inducer of this gene.

II-2) Assay method using the expression level of the present protein as an indicator The present invention provides a method for assaying glucose tolerance promoting activity, which comprises 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).

II-3) Assay method using reporter gene assay using expression control region of this gene The present invention comprises a method for assaying glucose tolerance promoting activity, comprising the following steps (1) to (3): provide. 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 this 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 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).
Insert the prepared reporter gene operably linked to the expression control region of this gene into a vector that can be used in the cell into which the reporter gene is to be introduced, using a conventional genetic engineering technique. And can be 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 II-1) to II-3), “cells capable of expressing the gene encoding the protein”, “cells capable of expressing the protein” or “expression control regions of the gene are linked in a functional manner” The cell containing the reporter gene thus formed 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, Further, it can be cultured in a commercially available medium such as D-MEM, OPTI-MEM, RPMI1640 medium (Gibco-BRL) and the like appropriately added with serum derived from mammals such as fetal bovine serum.

  In the above II-1) to II-3), “control cells not to be contacted with a test substance” means “cells capable of expressing a gene encoding this protein” and “expressing this protein” used in each first step. “Possible cell” or “cell containing a reporter gene operably linked to the expression control region of this gene” represents 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 II-1) to II-3), a test substance having an activity of inducing expression of the gene or the protein based on the expression level of the gene, the protein or the reporter gene measured in the first step and the second step. Can be selected. 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 inducer (expression promoting substance) of the present gene or protein.
The gene inducer or the expression inducer of the present protein selected as described above also has a glucose tolerance promoting activity, that is, an activity to improve glucose tolerance, more specifically, a treatment for a glucose metabolism-related disease of the present invention having an insulin response promoting activity. It is a candidate substance for an agent or prophylactic agent.

  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 dosage form and route of administration 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 administration form such as an injection, intravenously, artery, 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.

(Cloning of this gene from rat)
Rat Adipose cDNA Marathon-Ready cDNA (CLONTECH) 1 μL, Primer 1 20 pmol consisting of the base sequence shown in SEQ ID NO: 7, Primer 2 20 pmol consisting of the base sequence shown in SEQ ID NO: 8, TaKaRa Ex-Taq polymerase (Takara Shuzo) ) 50 μL of a reaction solution containing 5 μL of buffer attached to 2U and TaKaRa Ex-Taq polymerase and 4 μL of dNTP mixture (2.5 mM) attached to TaKaRa Ex-Taq polymerase was prepared. The PCR was performed under the condition that a heat retention cycle consisting of 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, 72 ° C. for 1 minute was repeated 50 times, and finally 72 ° C. for 5 minutes. After PCR, a PCR product showing about 1.5 Kbp was collected by agarose electrophoresis. Subsequently, the recovered PCR product was subcloned into pT7-Blue vector (Novagen), and then E. coli JM109 strain competent cell (Toyobo) was transformed with the plasmid. The rat-derived gene (rMO64) was isolated and purified from cultured cells obtained by culturing transformed cells in 100 mL of LB medium containing 50 μg / mL ampicillin using the QIAGEN Plasmid Maxi kit (QIAGEN). ) Was obtained.

(Determination of the base sequence of this rat-derived gene (rMO64))
Using the plasmid containing the PCR product (about 1.5 Kbp) obtained in Example 1 as a template, Thermo Sequenase II dye terminator kit (Amersham Pharmacia Biotech) and ABI373 DNA sequence reader (PE Applied Biosystems) According to the method of Sanger (F. Sanger, S. Nicklen, ARCoulson, Proceedings of National Academy of Science USA (1977), 74, 5463-5467), the rat-derived gene comprising the nucleotide sequence represented by SEQ ID NO: 3 The base sequence was determined.

(Cloning of this gene from mouse)
Mouse Normal Adipose cDNA (BioChain) 1 μL, Primer 3 20 pmol consisting of the base sequence shown in SEQ ID NO: 9, Primer 4 20 pmol consisting of the base sequence shown in SEQ ID NO: 10, TaKaRa Ex-Taq polymerase (Takara Shuzo) 2U, TaKaRa A 50 μL reaction solution containing 5 μL of buffer attached to Ex-Taq polymerase and 4 μL of dNTP mixture (2.5 mM) attached to TaKaRa Ex-Taq polymerase was prepared. PCR was performed under the condition that a heat retention cycle consisting of 94 ° C. for 30 seconds, 65 ° C. for 30 seconds, 72 ° C. for 2 minutes was repeated 50 times, and finally 72 ° C. for 5 minutes. After PCR, a PCR product showing about 1.2 kbp was collected by agarose electrophoresis. After the recovered PCR product was subcloned into pT7-Blue vector (Novagen), E. coli JM109 strain competent cell (Toyobo) was transformed with the plasmid. The mouse-derived gene (mMO64) is isolated and purified from cultured cells obtained by culturing the transformed cells in 100 mL of LB medium containing 50 μg / mL ampicillin using the QIAGEN Plasmid Maxi kit (QIAGEN). ) Was obtained.

(Determination of the base sequence of this mouse-derived gene)
Using the plasmid containing the PCR product (about 1.5 Kbp) obtained in Example 3 as a template, Thermo Sequenase II dye terminator kit (Amersham Pharmacia Biotech) and ABI373 DNA sequence reader (PE Applied Biosystems) This mouse-derived gene consisting of the nucleotide sequence shown in SEQ ID NO: 5 by the method of Sanger (F. Sanger, S. Nicklen, ARCoulson, Proceedings of National Academy of Science USA (1977), 74, 5463-5467) The nucleotide sequence of mMO64) was determined.

(Cloning of this human-derived gene)
Human Adipocyte Marathon-Ready cDNA (CLONTECH) 1 μL, Primer 5 20 pmol consisting of the base sequence shown by SEQ ID NO: 11, Primer 6 20 pmol consisting of the base sequence shown by SEQ ID NO: 12, TaKaRa Ex-Taq polymerase (Takara Shuzo) 2U Then, 50 μL of a reaction solution containing 5 μL of buffer attached to TaKaRa Ex-Taq polymerase and 4 μL of dNTP mixture (2.5 mM) attached to TaKaRa Ex-Taq polymerase was prepared. PCR was performed under the condition that a heat retention cycle consisting of 94 ° C. for 30 seconds, 65 ° C. for 30 seconds, 72 ° C. for 2 minutes was repeated 50 times, and finally 72 ° C. for 5 minutes. After PCR, a PCR product showing about 1.2 kbp was collected by agarose electrophoresis. After the recovered PCR product was subcloned into pT7-Blue vector (Novagen), E. coli JM109 strain competent cell (Toyobo) was transformed with the plasmid. This human-derived gene (hMO64) is obtained by isolating and purifying the transformed cells from cultured cells obtained by culturing in 100 mL of LB medium containing 50 μg / mL ampicillin using QIAGEN Plasmid Maxi kit (QIAGEN). ) Was obtained.

(Determination of the base sequence of this human-derived gene)
Using the plasmid containing the PCR product (about 1.5 Kbp) obtained in Example 5 as a template, Thermo Sequenase II dye terminator kit (Amersham Pharmacia Biotech) and ABI373 DNA sequence reader (PE Applied Biosystems) According to the method of Sanger (F. Sanger, S. Nicklen, ARCoulson, Proceedings of National Academy of Science USA (1977), 74, 5463-5467), the human-derived gene comprising the nucleotide sequence represented by SEQ ID NO: 1 The base sequence was determined.

(Construction of a vector for E. coli expression of this human-derived gene)
0.1 μg of plasmid containing the human-derived gene obtained in Example 6, 20 pmol of primer 7 consisting of the base sequence shown in SEQ ID NO: 13, 20 pmol of primer 8 consisting of the base sequence shown in SEQ ID NO: 14, TaKaRa Ex-Taq A 50 μL reaction solution containing 5 μL of polymerase (Takara Shuzo Co., Ltd.) 2 U, 5 μL of buffer attached to TaKaRa Ex-Taq polymerase and 4 μL of dNTP mixture (2.5 mM) attached to TaKaRa Ex-Taq polymerase was prepared. PCR was performed under the condition that a heat retention cycle consisting of 94 ° C. for 30 seconds, 65 ° C. for 30 seconds, 72 ° C. for 2 minutes was repeated 50 times, and finally 72 ° C. for 5 minutes. The PCR product was digested with restriction enzymes NdeI and BamHI, and a PCR product showing about 1.2 kbp was recovered by agarose electrophoresis. The recovered PCR product was cloned into pET16b (Novagen) and then transformed into E. coli BL21 (DE3) plysS (Novagen) according to the usual method.

(Purification of this human-derived protein)
Recombinant E. coli (hMO64-pET16b / BL21) prepared in Example 7 was added to 30 mL of LB medium (trypton 1 w / v%, yeast extract 0.5 w / v%, NaCl 0.5 w / v%) containing 50 ug / ml of ampicillin. After inoculation, the cells were cultured overnight at 37 ° C with shaking. After inoculating 15 mL of the culture solution after culture to 500 mL of LB medium (trypton 1 w / v%, yeast extract 0.5 w / v%, NaCl 0.5 w / v%) containing ampicillin 50 ug / ml, after culturing at 37 ° C. for 2 hours, IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 0.5 mM, and the cells were further cultured at 37 ° C. for 4 hours. The cells were collected by centrifugation (8000 × g, 5 minutes, 4 ° C.), and the cells were sonicated and then centrifuged (12000 × g, 15 minutes, 4 ° C.) to obtain the supernatant. 10 mL of the resulting supernatant was passed through a column packed with 1 mL of nickel-bound affinity carrier (Ni-NTA, manufactured by QIAGEN). The column was washed with a washing solution containing 25 mM imidazole (25 mM imidazole, 50 mM Tris pH 8.0) and then eluted with an eluent containing 400 mM imidazole (400 mM imidazole, 50 mM Tris pH 8.0) to obtain an affinity purified MO64 protein. Affinity-purified protein MO64 is replaced with 20 mM Tris pH 7.0 solution, adsorbed on a cation exchange chromatography column (S-Sepharose, Amersham Pharmacia), and the salt concentration gradient is increased with 20 mM Tris pH 7.0, 1 M NaCl. To obtain the final purified hMO64 protein.

(Creation of DIO (dietary obesity induction) mice)
Individual ICR mice are divided into a normal diet group (Std diet group) and a high-fat / high-sugar diet group (HH diet group) so that the weight average values are about the same. Ordinary food (D12328 (manufactured by Research Diets)) or high-fat food (D12331 (manufactured by Research Diets)) was freely consumed. Environmental conditions for breeding are made of stainless steel in a breeding room maintained at room temperature 20-26 ° C, humidity 40-70%, light and darkness for 12 hours each (lighting: 6 am to 6 pm), ventilation rate 12 times / hour Individual breeding was performed in a six-cage cage (W: 750 x D: 100 x H: 100 mm).

(Administration experiment 1 of human-derived protein to obese animals)
One group of 10 DIO (dietary obesity-inducing) mice (male) constructed by the method described in Example 9 was used as one group. To this, a PBS buffer solution of the human-derived protein prepared in Example 8 was intraperitoneally administered for 2 weeks so that the pure amount of the test substance was 1 mg / kg-body weight x 2 times / day. Two weeks after administration, a glucose tolerance test and an insulin responsiveness test were performed.
The glucose tolerance test and the insulin responsiveness test were performed by the following methods.
(1) Glucose tolerance test (OGTT): A glucose solution (1 g / kg, 10 mL / kg) was given to animals that had been fasted for 18 to 24 hours under water intake from the previous day after the long-term dietary loading shown in Example 9 Was orally administered (a disposable syringe made of polypropylene equipped with a disposable metallic sonde). Approximately 50 μL of blood was collected from the orbital venous plexus of the animal using heparinized capillaries before glucose administration and 20, 60 and 120 minutes after glucose administration, and an oxygen electrode method using Antsense II (Daikin Industries, Ltd.) The blood glucose level of whole blood was measured.
(2) Insulin tolerance test (ipITT): After the above OGTT, the test was performed at intervals of about 2 weeks. The insulin solution (0.35 U / kg, 10 mL / kg) was intraperitoneally administered to animals that had been fasted for 18 to 24 hours under water intake from the previous day (a polypropylene disposable syringe equipped with an injection needle). The blood glucose level was measured by the above method (1) immediately before insulin loading, 30, 60 and 120 minutes later.

(result)
In the glucose tolerance test, the group of mice administered with the human-derived protein showed a significant hypoglycemic effect 120 minutes after glucose administration compared to the group administered with phosphate buffer (Table 1). In addition, when an insulin tolerance test was performed and compared in the same manner, the group of mice administered with the human-derived protein showed a significant hypoglycemic effect 60 minutes and 90 minutes after glucose administration compared to the phosphate buffer group. (Table 2). From these results, it was shown that glucose tolerance and insulin responsiveness were improved by administration of this protein to the obese mouse group (dietary obesity-inducing mice).

(Experimental administration of this gene to obese animals)
Administration of this gene to obese animals can be performed by adenovirus. Adenovirus is prepared using Adenovirus Expression Kit (Takara Shuzo).
i) Insertion of this gene into a cosmid vector The sense strand of the mouse MO64 gene prepared in Example 4 is inserted into the SwaI site of the cosmid vector pAxCAwt (Takara Shuzo) to obtain pAxCAwt-mMO64. The obtained pAxCAwt-mMO64 is in vitro packaged using λ packaging kit GigapackXL (Stratagene) and infected with E. coli DH5α. Infected E. coli is cultured in 50 ml 50 μg / ml ampicillin-containing LB medium, and a large amount of cosmid DNA is prepared by Lambda DNA purification Kit (Toyobo).
ii) Production of recombinant adenovirus
293 cells (Takara Shuzo) are cultured in 10% FCS-added D-MEM medium (Takara Shuzo) at 37 ° C under 5% carbon dioxide. 8 μg of pAxCAwt-mMO64 prepared in i) and 5 μl of restriction enzyme-treated DNA-TPC (Takara Shuzo) are mixed, and 293 cells are co-transfected by the calcium phosphate method using the mixture. The cells are cultured as they are for 18 hours, and then the medium is replaced with D-MEM medium supplemented with 10% FCS (Takara Shuzo) and further cultured for 12 hours. After culturing, the cells are removed from the dish, and the collected cell suspension and 293 cells are mixed and further cultured in D-MEM medium (Takara Shuzo) with 10% FCS. At the time of death, the culture is rapidly frozen with dry ice. After freezing and thawing 6 times, the supernatant obtained by centrifugation at 5000 rpm for 5 minutes is stored as a primary recombinant adenovirus solution (AxCAwt-mMO64).

iii) Confirmation and titration of recombinant adenovirus
Add 10 μl of the primary recombinant virus solution prepared in the previous section to 293 cells (Takara Shuzo), add 5% FCS-added D-MEM medium (Takara Shuzo), add the mixture to 37 ° C, 5% dioxide. Incubate for 1 hour under carbon. Further, 5% FCS-added D-MEM medium (Takara Shuzo) is added to the mixture, followed by culturing at 37 ° C. under 5% carbon dioxide. After culturing for 3 days, the culture solution of virus-infected 293 cells (Takara Shuzo) is collected and rapidly frozen on dry ice. After freezing and thawing six times, the supernatant obtained by centrifugation at 5000 rpm for 5 minutes is stored as a secondary recombinant adenovirus solution. On the other hand, the precipitate (cells) obtained simultaneously by the above operation is also collected and rapidly frozen with dry ice. To the collected cells, 40 μl of 10 × TNE (500 mM Tris-HCl (pH = 7.5), 1M NaCl, 100 mM EDTA), 4 μl of proteinase K (20 mg / ml), and 356 μl of sterilized distilled water were added, and the mixture was well suspended in Voltex. To the obtained suspension, 4 μl of 10% SDS is added, and this is incubated at 50 ° C. for 1 hour. Then, after phenol / chloroform / isoamyl alcohol (Nippon Gene) treatment, ethanol precipitation is performed, and the resulting DNA pellet is air-dried and then dissolved in 50 μl of sterile distilled water containing 20 μg / ml RNase A (Nippon Gene). Obtain a recombinant adenovirus DNA solution. The obtained recombinant adenovirus DNA solution is cleaved with XhoI 5U (Takara Shuzo) to confirm the target fragment (0.48 Kbp). Subsequently, this recombinant adenovirus DNA is subcultured by repeating the above procedure. In this way, a high-titer recombinant virus is produced. The titer is measured by an existing method (Japanese Patent Laid-Open No. 7-298877).

iv) Administration of this gene to obese animals To 10-week-old male KKAy mice (Claire Japan), which are obese animals, adenovirus dose of 1x10 ¹⁰ PFU (10 mice per group) Prepare by diluting the recombinant adenovirus prepared in the previous section with physiological saline. This aqueous solvent is administered intraperitoneally to the DIO (dietary obesity-inducing) mouse prepared in Example 9 using 0.2 ml of syringe per mouse. Two weeks after the administration, a glucose tolerance test and an insulin responsiveness test are performed on DIO mice, which are test obese animals.

(Production of transformed non-human animals: Production of transformed mice)
i) Preparation of transgene fragment The mouse MO64 gene prepared in Example 4 was transformed into a plasmid having a mouse aP2 promoter region (about 5.4 kb) (Shimomura, I. et al., Genes & Developmemt, 12, 3182 (1998). )) To obtain the plasmid pBlueSK / maP2pro / mMO64 / polyA.
The obtained pBlueSK / maP2pro / mMO64 / polyA is digested with HindIII and NotI, thereby obtaining about 5 μg of a fragment (about 7.1 kb) containing the mouse aP2 promoter region, the mouse-derived gene and SV40E polyA (hereinafter referred to as “a”). This is referred to as this transgene.) This transgene preparation solution is obtained by preparing this transgene with 10 mM Tris-HCl (pH 8.5) and 1 mM EDTA · 2Na (pH 8.5) to a concentration of 0.1 μg / μl.
ii) Harvesting of fertilized eggs Using C57BL / 6J female and male mice (8 weeks of age or older), superovulation is induced by intraperitoneal administration of 5 units of pregnant mare serum gonadotropin and human ciliary gonadotropin. Then, pronuclear fertilized eggs are obtained by in vitro fertilization. In order to remove cumulus cells and the like, the fertilized eggs were prepared using mWM medium (5.14 g / L NaCl, 0.36 g / L KCl, 0.16 g / L KH ₂ PO ₄ , 0.53 g / L lactate Ca · _{5H 2 O, 0.29g / L MgSO} 4 / 7H 2 O, 0.19g / L NaHCO 3, 37mg / L EDTA / 2Na, 1g / L glucose, 3.7 ml / L-lactic acid Na, 35 mg / L pyruvate Na 3 g / L bovine serum albumin, 80 mg / L penicillin, 50 mg / L streptomycin, 5 mg / L phenol red).
iii) Injection of this transgene into fertilized eggs The microinjection system is composed of a micromanipulator device (manufactured by Leica) and an Hoffman inverted microscope (manufactured by Zeiss). The transgene preparation solution described in the previous section is prepared at about 500 to about 1000 copies / μl, and the supernatant obtained by centrifuging at 13,000 rpm for 3 minutes is used as the transgene solution. In the injection chamber of the micromanipulator, the fertilized egg described in the preceding section ii) is fixed with a support pipette, and about 2 μl of the above-mentioned transgene preparation solution placed in the injection pipette is injected into the male pronucleus. After the injection, the cells are cultured in mWM medium in the presence of 5% CO ₂ at 37 ° C. for 12 to 16 hours. Embryos that have developed at the 2-cell stage are transferred to the fallopian oviduct.
iv) Transplantation of fertilized eggs into temporary parents 20-25 embryos per mouse are injected into the left and right oviducts of syngeneic pseudopregnant female mice prepared in advance for transplantation.
v) Selection of transformed mice Pups are obtained after about 3 weeks from female mice transplanted with fertilized eggs. When the pup is about 6 weeks old, a part of the tail is cut out to extract chromosomal DNA, and the presence of a fragment derived from the transgene in the extracted DNA is examined by PCR. Chromosomal DNA is described in the usual methods (for example, Sambrook J., Frisch EF, Maniatis T., Molecular Cloning 2nd edition), Cold Spring Harbor Laboratory publication (Cold Spring Harbor Laboratory press), etc. Method). Using 5 ng of the obtained chromosomal DNA as a template, 10 pmol each of DNA consisting of the nucleotide sequences shown in SEQ ID NO: 15 and SEQ ID NO: 16 as primers, and using ExTaq polymerase 2.5 units (manufactured by Takara Shuzo) at 94 ° C for 2 minutes, Incubate for 1 cycle at 58 ° C for 2 minutes and 72 ° C for 2 minutes, then 94 ° C for 1 minute, 58 ° C for 1 minute, and 72 ° C for 1 minute. The PCR reaction is performed under the condition of incubation at the end of the cycle for 5 minutes at 72 ° C. The PCR reaction product obtained by the reaction is subjected to agarose gel electrophoresis. An individual whose target fragment has been confirmed is judged to be a transformed mouse in which the present transgene is inserted into the chromosome (hereinafter referred to as the present transformed mouse).
vi) Analysis of copy number of this transgene Next, the copy number of this transgene in this transformed mouse was analyzed by the usual Southern blotting method (eg, Sambrook J., Frisch EF, Maniatis T., Molecular Cloning 2nd Edition). (Molecular Cloning 2nd edition), the method described in Cold Spring Harbor Laboratory press). The plasmid pBlueSK / maP2pro / mMO64 / polyA obtained in the previous section is digested with BamHI. The obtained about 0.7 Kb fragment was labeled with ³² P using a commercially available radiolabeling kit (Random Prime Labeling Kit, manufactured by Boehringer Mannheim) according to the attached protocol, and used as a detection probe (hereinafter referred to as this detection probe). .) The detection probe is used after being heated at 100 ° C. for 10 minutes and then rapidly cooled. Chromosomal DNA extracted according to the method described in the previous section is cleaved with a restriction enzyme such as EcoRI, BamHI, HindIII, and subjected to agarose gel electrophoresis. The gel after electrophoresis was washed with a solution containing 1.5 M NaCl and 0.15 M trisodium citrate (hereinafter referred to as 10 × SSC), and then a capillary was put on a commercially available nylon membrane Hybond-N (manufactured by Amersham Pharmacia). Blot by method. The membrane is put in a polyethylene bag, a DIG EASY Hyb solution (manufactured by Boehringer Mannheim) to which denatured salmon sperm DNA is added to a concentration of 100 μg / ml is added and incubated at 50 ° C. for 2 hours. The solution in the bag is removed, and 2 to 3 ml of DIG EASY Hyb solution to which denatured salmon sperm DNA is newly added to a concentration of 100 μg / ml is injected. Further, 1 ml or less of this detection probe is added, and kept at 50 ° C. for about 15 hours. After the incubation, the membrane is taken out and incubated at room temperature for 15 minutes in a solution of 10 × SSC diluted 5-fold containing 0.1% sodium lauryl sulfate (hereinafter referred to as SDS). The operation of taking out the membrane and keeping it warm in a solution obtained by diluting fresh 10 × SSC 5 times is further repeated once or twice. Furthermore, it is kept at 50 to 68 ° C. for 30 minutes in a solution obtained by diluting 10 × SSC 100 times containing 0.1% SDS. After taking out the membrane and removing excess water, the imaging plate (BAS3-2040, manufactured by Fuji Film) is exposed. The exposed imaging plate is analyzed with an image analyzer (manufactured by Fujifilm), and the signal on the membrane is detected.
The signal on the membrane is detected by the Southern blotting method as described above except that the chromosomal DNA of a mouse into which this transgene has not been introduced (hereinafter referred to as the present wild type mouse) is used. The signal of this transformed mouse and this wild type mouse are compared, and the number of introduced copies of this gene is evaluated from the intensity of the signal detected only in this transformed mouse.

SEQ ID NO: 7: PCR primer SEQ ID NO: 8: PCR primer SEQ ID NO: 9: PCR primer SEQ ID NO: 10: PCR primer SEQ ID NO: 11: PCR primer SEQ ID NO: 12: PCR primer SEQ ID NO: 13: PCR primer sequence Number 14: PCR primer

Claims (9)

They are selected from any of (a) ~ (f) below, as an active ingredient a 蛋 white matter, sugar metabolism-related diseases or preventive agent:
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2,
In the amino acid sequence represented by (b) SEQ ID NO: 2, one or more amino acids are deleted, added, inserted or substituted amino acid sequence, and a protein having a glucose tolerance improving activity,
(C) SEQ ID NO: from the 22 th amino acid in the amino acid sequence represented by 2 comprising the amino acid sequence represented by the 414 amino acid protein,
(D) a protein comprising an amino acid sequence obtained by adding methionine to the amino terminus of the amino acid sequence of (c),
(E) a protein comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2 and having a glucose tolerance improving activity ,
(F) a protein comprising an amino acid sequence encoded by a DNA having a base sequence represented by nucleotides from the 224th nucleotide to the 1465th nucleotide in the base sequence represented by SEQ ID NO: 1,
(G) encoded by DNA having a base sequence having 80% or more sequence identity with the DNA having the base sequence shown by nucleotides 224 to 1465 in the base sequence shown by SEQ ID NO: 1 that to the amino acid sequence, and proteins that have a glucose tolerance improving activity,
(H) It hybridizes under stringent conditions with DNA having complementarity to DNA having the nucleotide sequence represented by nucleotides 224 to 1465 in the nucleotide sequence represented by SEQ ID NO: 1. It consists encoded amino acid sequence by DNA, and proteins that have a glucose tolerance improving activity. Therapeutic or prophylactic agent for the content to, glucose metabolism disorders, as an active ingredient a gene encoding 蛋 white matter according to any one of claims 1 (a) ~ (h) . Characterized by having a insulin responsiveness promoting activity, therapeutic or prophylactic agent according to claim 1 or 2. The glucose tolerance-improving activity of the test substance, using as an index whether the test substance promotes the expression of the protein according to any one of (a) to (h) or the gene encoding the protein. Test method. The method for assaying glucose tolerance improving activity according to claim 4, comprising the following steps (1) to (3):
(1) A first step of contacting a test substance with a cell capable of expressing a gene encoding the protein according to any one of (a) to (h) of claim 1 ,
(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 glucose tolerance improving 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 step. The method for assaying glucose tolerance improving activity according to claim 4, comprising the following steps (1) to (3):
(1) a first step of bringing a test substance into contact with a cell capable of expressing the protein according to any one of (a) to (h) of claim 1 ;
(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 glucose tolerance improving 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 process. The method for assaying glucose tolerance improving activity according to claim 4, comprising the following steps (1) to (3):
(1) a test substance and a cell containing a reporter gene operatively linked to an expression control region of the gene encoding the protein according to any one of (a) to (h) The first step of contacting,
(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;
(3) A third step of evaluating the glucose tolerance improving 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 4 to 7, wherein the glucose tolerance improving activity is insulin responsiveness promoting activity. A candidate for a therapeutic or prophylactic agent for a glucose metabolism-related disease is selected using the glucose tolerance improving activity of the test substance as an index measured by the assay method according to any one of claims 4 to 7. And a method for searching for a therapeutic or preventive agent for a sugar metabolism-related disease .