JP2008239518A - Agent for prevention and treatment of obesity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an agent for improving sugar and lipid metabolism, a method for screening a substance effective for the improvement of sugar and lipid metabolism, a non-human transgenic animal useful in the screening method, and a fat tissue or a fat cell derived from the non-human transgenic animal. <P>SOLUTION: The invention provides an agent for improving sugar and lipid metabolic ability (especially an agent for ameliorating a fat state), containing known membrane protein OL16 called also as ACAM (Adipocyte adhesion molecule) and expressing in a white fat cell or a substance to promote the expression of OL16 as an active component; a method for assaying sugar and lipid metabolic ability by using the expression quantity of OL16 or OL16 gene as an index; a non-human transgenic animal (especially a model animal of antiobesic or antidiabetic state) having a fat cell-specifically introduced OL16 gene; and a fat tissue or a fat cell derived from the non-human transgenic animal. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention provides an agent for improving sugar / lipid metabolism, a method for screening a substance effective for improving sugar / lipid metabolism, a transgenic non-human animal useful for the screening method, and the like.

Obesity is a condition in which the body weight has increased beyond the standard body weight as a result of abnormal accumulation of neutral fat in adipose tissue, particularly subcutaneous adipose tissue. In particular, visceral adipose tissue that accumulates around the viscera such as the mesentery is considered to cause abnormal metabolism of sugars and lipids, such as arteriosclerosis, diabetes, and diseases such as myocardial infarction and cerebral infarction (these diseases Collectively, it is sometimes called lifestyle-related diseases or metabolic syndrome). For this reason, many studies have been conducted on the relationship between fat cells and sugar / lipid metabolism.
On the other hand, OL16 is a protein having homology with cell adhesion molecules such as ICAM, and its amino acid sequence and gene sequence encoding OL16 are known (Genbank accession number: NM_133733; see Non-Patent Document 1). . OL16, also called ACAM (Adipocyte adhesion molecule), is a membrane protein expressed in white adipocytes, and is known to have an immunoglobulin domain in the extracellular domain. However, the function of OL16 in vivo is unknown, and the relationship with the ability of sugar / lipid metabolism has not been known at all.
Eguchi et al Biochemical Journal Vol.387 343-353 2005

  The problem to be solved by the present invention is to provide a therapeutic agent or prophylactic agent for a disease accompanied by abnormal sugar / lipid metabolism having a new mechanism of action, a method for searching for the therapeutic agent or prophylactic agent, and the like.

  As a result of analyzing a gene expressed in an obese model animal, the present inventors have found that the expression of OL16 is remarkably increased. In order to elucidate the role of OL16 in the obesity state, the present inventors produced a transgenic animal that expresses OL16 specifically in adipocytes, and analyzed its phenotype. As a result, the transgenic animals introduced with the prepared OL16 have reduced body weight gain, superior glucose tolerance, and increased energy consumption compared to control animals even under high fat diets. I understood. That is, the present inventors have found that OL16 acts as a factor that can improve an individual's ability to metabolize sugar and lipid in adipocytes, and have completed the present invention.

That is, the present invention
[1] An agent for improving sugar / lipid metabolism, comprising OL16 or a substance that promotes the expression of OL16 as an active ingredient;
[2] The agent for improving sugar / lipid metabolism ability according to [1], wherein the substance that promotes the expression of OL16 is a gene encoding OL16;
[3] The agent for improving sugar / lipid metabolism ability according to [1], wherein the substance that promotes the expression of OL16 is an expression vector containing a gene encoding OL16;
[4] The sugar / lipid metabolic ability improving agent according to any one of [1] to [3], which is used as a preventive and therapeutic agent for a disease caused by impaired glucose tolerance or a disease caused by obesity;
[5] The sugar / lipid metabolic ability improving agent according to any one of [1] to [3], which is used as an agent for improving obesity.
[6] A method for assaying the ability of a test substance to metabolize sugar and lipid, using as an index whether or not the test substance promotes the expression of OL16 or a gene encoded by OL16;
[7] The assay method according to [6], 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 OL16;
(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 ability of the test substance to metabolize sugar / lipid 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;
[8] The assay method according to [6], comprising the following steps (1) to (3):
(1) a first step of contacting a test substance with a cell capable of expressing OL16;
(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 ability of the test substance to metabolize sugar and lipid 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;
[9] The assay method according to [6], 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 that is operably linked to an expression control region of a gene encoding OL16;
(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 sugar / lipid metabolic ability 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;
[10] The assay method according to [6], comprising the following steps (1) to (3):
(1) A step of administering a test substance to an animal with abnormal sugar / lipid metabolism,
(2) a step of measuring the expression level of OL16 or OL16 gene in an animal administered with the test substance and comparing it with the expression level in a control animal not administered with the test substance;
(3) A third step of evaluating the sugar / lipid metabolic capacity of the test substance using as an index that the expression level of OL16 or OL16 gene is larger than the expression level in the control animal based on the comparison result of the second step;
[11] The assay method according to [10], wherein the animal with abnormal sugar / lipid metabolism is a high-fat diet-fed animal;
[12] The assay method according to [10] or [11], wherein the animal with abnormal sugar / lipid metabolism is a mouse or a rat;
[13] Selecting candidate substances for improving sugar / lipid metabolism ability using as an index the sugar / lipid metabolism ability of the test substance measured by the assay method according to any one of [6] to [12] A method for searching for an agent for improving sugar / lipid metabolism ability,
[14] An agent for improving sugar / lipid metabolism, comprising a substance selected by the search method according to [13] or a pharmaceutically acceptable salt thereof as an active ingredient;
[15] The agent for improving sugar / lipid metabolism ability according to [14], which is used as a preventive and therapeutic agent for a disease caused by impaired glucose tolerance or a disease caused by obesity;
[16] The agent for improving sugar / lipid metabolic ability according to [14], which is used as an agent for improving obesity;
[17] A transgenic non-human animal into which the OL16 gene has been introduced, characterized in that OL16 is expressed specifically in adipose tissue;
[18] The transgenic non-human animal according to [17], which is a model animal for anti-obesity or anti-diabetes;
[19] Adipose tissue or adipocytes derived from the transgenic non-human animal according to [17];
About.

  According to the present invention, an agent for improving sugar / lipid metabolic ability, more specifically, a therapeutic agent for diseases caused by impaired glucose tolerance or diseases caused by obesity (eg, lifestyle diseases such as hyperlipidemia, hypertension). Alternatively, it has become possible to provide a prophylactic agent, an agent for improving obesity, a method for assaying the ability to metabolize sugar and lipid, a method for searching for a drug using the assay method, and the like.

The present invention is described in detail below.
(I) Sugar / lipid metabolic capacity improving agent The first aspect of the present invention relates to a sugar / lipid metabolic capacity improving agent containing OL16 or a gene encoding OL16 as an active ingredient.
In this specification, OL16 is a known protein also called ACAM (Adipocyte adhesion molecule), and human OL16 (SEQ ID NO: 2) and its gene sequence (SEQ ID NO: 1) are known as Genbank accession number: NM_024769. Yes. In this specification, OL16 is a concept including not only human OL16 but also orthologs such as homologues derived from other animal species, mutants thereof (including SNPs and haplotypes), and the like. The orthologue of OL16 is not particularly limited, and can be derived from animals such as mammals (eg, cows, sheep, goats, monkeys, rabbits, rats, hamsters, guinea pigs, mice).
That is, examples of OL16 include the following amino acid sequence groups (hereinafter sometimes referred to as the present protein).

<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;
(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 360th nucleotide to the 1478th 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 360 to 1478 in the base sequence shown by SEQ ID NO: 1. And an amino acid sequence having the ability to improve sugar / lipid metabolism,
(H) It hybridizes under stringent conditions with DNA having complementarity to DNA having the nucleotide sequence represented by nucleotides from the 360th nucleotide to the 1478th nucleotide in the nucleotide sequence represented by SEQ ID NO: 1. An amino acid sequence encoded by DNA and having an ability to improve sugar / lipid metabolism.

Examples of the “amino acid deletion, addition, insertion or substitution” in (b) and “80% or more sequence identity” in (e) and (g) include, for example, the amino acid sequence represented by SEQ ID NO: 2 This includes a naturally occurring mutation due to the processing that a protein having a protein undergoes in a cell, the species difference of an organism from which the protein is derived, an individual difference, a difference between tissues, and the like, 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, 1 or several, 1 to 10, or more. The number of such modifications may be in a range in which the ability of the protein to improve sugar / lipid metabolism 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.

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 above-mentioned protein include a rat-type homolog (Genbank accession number: NM_173154) and a mouse-type homolog (Genbank accession number: NM_133733) in addition to the human protein consisting of the amino acid sequence represented by SEQ ID NO: 2. Can be mentioned.

In the present specification, examples of the gene encoding OL16 (the present protein) include the following base sequence groups (hereinafter sometimes referred to as the present gene).
<Base sequence group>
(I) a base sequence encoding the amino acid sequence represented by SEQ ID NO: 2,
(J) a base sequence encoding 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;
(M) a base sequence that encodes an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, and wherein the protein comprising the amino acid sequence has an ability to improve sugar / lipid metabolism;
(N) Complementary to DNA having the base sequence represented by SEQ ID NO: 1, or (q) the nucleotide sequence represented by nucleotides 360 to 1478 in the base sequence represented by SEQ ID NO: 1. A DNA base sequence that hybridizes with stringent DNA under stringent conditions and the protein encoded by the base sequence has an ability to improve sugar / lipid metabolism,
A gene having a base sequence such as

The preparation method of this gene or this protein is demonstrated below.
The above-mentioned gene encoding OL16 (this gene) is converted into a conventional genetic engineering method (eg, Sambrook J., Frisch EF, Maniatis T., Molecular Cloning 2nd edition), Cold Spring Harbor Laboratory. (According to the method published in 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 Bio and the like. Restriction enzymes used for introducing a gene encoding this protein into the expression vector are also commercially available from Takara Bio and others. 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 Bio.
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, “sugar / lipid metabolism improving ability” is characterized by having glucose tolerance enhancing activity and insulin responsiveness promoting activity, ie, glucose tolerance improving activity. Specifically, it represents an activity of lowering blood glucose level in vivo in mammals, an activity of reducing adipose tissue weight or body weight, and / or an activity of enhancing energy consumption (lipid metabolism).
That is, the ability of OL16 to improve sugar / lipid metabolism can be confirmed by a decrease in blood glucose level in the animal when administered to a high-fat loaded animal (such as a mouse or rat). Specifically, the ability to improve glucose metabolism can be confirmed 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). . That is, it can be said that sugar metabolism is improved if the decrease in blood glucose level after sugar loading is enhanced in OGTT. For the implementation method of OGTT, a biochemical experiment book (Daiichi Shuppan) may be referred to.

In the present specification, the “disease caused by impaired glucose tolerance” includes a disease accompanied by an abnormal glucose metabolism function in a living body, specifically, type 2 diabetes, diabetic retinopathy, diabetic nephropathy, Examples include diabetic neuropathy, or diseases such as diabetes, arteriosclerosis associated with persistent glucose tolerance or hyperglycemia. That is, the disease caused by impaired glucose tolerance is not particularly limited as long as it is a disease having a pathological condition caused by impaired blood glucose regulation such as decreased 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 or prophylactic agent for a glucose 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. The term “disease” refers to a condition in which symptoms are exacerbated by obesity such as the accumulation of visceral fat, and specifically includes lifestyle diseases such as hyperlipidemia or hypertension, hypertriglyceridemia and hypercholesterolemia. Can be mentioned.
In the present specification, “improvement of obesity” means a decrease in body weight, preferably a decrease in the amount of fat accumulated in a living body, more preferably a decrease in visceral fat.

  The agent for improving the ability to metabolize sugar and lipid of the present invention is not limited to a drug such as a therapeutic or preventive agent for a disease caused by abnormal glucose tolerance or a disease caused by obesity, but also for in vivo or in vitro biological activity measurement. -It is also useful as a research reagent for enhancing lipid metabolic capacity.

  In the present specification, the “substance that promotes the expression of OL16” is not particularly limited, and examples thereof include a gene encoding OL16. The method for preparing the gene is as described above.

The gene encoding OL16 can also be administered to a living body as a normal expression vector (hereinafter referred to as OL16 expression vector). In the OL16 expression vector, the above-mentioned polynucleotide encoding OL16 must be operably linked to a promoter capable of exhibiting promoter activity in mammalian cells to be administered. The promoter used is not particularly limited as long as it can function in the mammal to which it is administered. For example, SV40-derived early promoter, cytomegalovirus LTR, Rous sarcoma virus LTR, MoMuLV-derived LTR, adenovirus-derived Examples include viral promoters such as early promoters, and mammalian constituent protein gene promoters such as β-actin gene promoter, PGK gene promoter, transferrin gene promoter, and the like. In addition, a promoter specific for OL16-expressing cells in vivo such as fat cells may be used. Such a promoter can be a promoter of an arbitrary gene that is specifically expressed in OL16-expressing cells. For example, as a fat cell-specific promoter, an aP2 (fatty acid binding protein) promoter, adiponectin (adiponectin) A promoter, an adipsin promoter, or the like can be used.
The OL16 expression vector preferably contains a transcription termination signal, that is, a terminator region, downstream of the oligo (poly) nucleotide encoding the nucleic acid molecule. In addition, selectable marker genes for selection of transformed cells (such as genes that confer resistance to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, phosphinothricin, genes that complement auxotrophic mutations, etc.) You can also
The basic skeleton vector used as the expression vector is not particularly limited, but suitable vectors for administration to mammals such as humans include adenovirus, retrovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, Examples thereof include viral vectors such as poliovirus, Sindbis virus and Sendai virus.

(II) Assay method The second aspect of the present invention is the sugar / lipid metabolism of the test substance, which uses whether or not the test substance promotes the expression of the present protein or a gene encoding the present protein (this gene). It relates to a test method for improvement ability.
(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 ability of the test substance to improve sugar / lipid metabolism 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 such as adipocytes, 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, hamsters, and guinea pigs, rabbits, 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 cells are not particularly limited as long as they potentially express OL16. It may be a cell that constitutively expresses OL16, a cell that expresses OL16 under induction conditions (eg, treatment with a drug), and the like. Such cells can be easily obtained by those skilled in the art, and primary cultured cells, cell lines derived from the primary cultured cells, commercially available cell lines, cell lines available from cell banks, and the like can be used. For example, 3T3L1 cells, 3T3-F442A cells, C3H10T1 / 2 cells and the like can be mentioned. In addition, cells derived from a model animal with abnormal sugar / lipid metabolism described below (eg, primary cultured cells) may be used.
The compound used as a test substance is not particularly limited, and is a protein, peptide, amino acid, nucleic acid (eg, nucleiside, oligonucleoside, polynucleoside), carbohydrate (eg, monosaccharide, disaccharide, oligosaccharide, polysaccharide), lipid (Eg, saturated or unsaturated linear, branched and / or cyclic fatty acids), inorganic compounds, naturally occurring organic compounds (compounds derived from microorganisms, animals and plants, marine organisms, etc.) 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.

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.

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).

In the step (1), the contact of the test substance with the cells can be performed in the medium by a method known per se. The culture medium in which the test substance is contacted with cells capable of measuring the expression of OL16 is appropriately selected according to the type of cells used, for example, a minimum essential amount containing about 5 to 20% fetal calf serum. Medium (MEM), Dulbecco's modified minimal essential medium (DMEM), RPMI 1640 medium, 199 medium, and the like. The culture conditions are also appropriately determined according to the type of cells used, etc. For example, the pH of the medium is about 6 to about 8, the culture temperature is usually about 30 to about 40 ° C., and the culture time is About 12 to about 72 hours. In the step (1), first, the expression level of OL16 in the cell contacted with the test substance is measured. The expression level can be measured by a method known per se in consideration of the type of cells used. For example, when an OL16-expressing cell is used as a cell capable of measuring OL16 expression, the expression level is measured by, for example, preparing total RNA from the cell, RT-PCR, Northern blotting, etc. Can be done. The expression level of the translation product can be measured by preparing an extract from the cells and using an immunological technique. As an immunological technique, a radioisotope immunoassay (RIA method), an ELISA method (Methods in Enzymol. 70: 419-439 (1980)), a fluorescent antibody method, or the like can be used. On the other hand, when a cell capable of measuring reporter assay for the transcriptional regulatory region is used as a cell capable of measuring OL16 expression, the expression level can be measured based on the signal intensity of the reporter. Next, the expression level of OL16 in the cells contacted with the test substance is compared with the expression level of OL16 in the control cells not contacted with the test substance. The comparison of expression levels is preferably performed based on the presence or absence of a significant difference. The expression level of OL16 in the control cells not contacted with the test substance is the expression level measured at the same time, even if the expression level was measured in advance with respect to the measurement of the expression level of OL16 in the cells contacted with the test substance. However, the expression level is preferably measured simultaneously from the viewpoint of the accuracy and reproducibility of the experiment.
When using Northern blotting, the primer or probe is labeled with a radioisotope (32P, 33P, etc .: RI) or a fluorescent substance and hybridized with cell-derived RNA transferred to a nylon membrane or the like according to a conventional method. After soy, the formed duplex of the primer or probe (DNA or RNA) and RNA is used as a signal derived from the primer or probe label (RI or fluorescent substance) as a radiation detector (BAS- 1800II (manufactured by Fuji Film) or a method of detecting and measuring with 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 sugar / lipid metabolic capacity, 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 ability of the test substance to improve sugar / lipid metabolism 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.

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 the primary antibody, and then binds to the primary antibody labeled with a radioactive isotope such as 125I, a fluorescent substance, or an enzyme such as horseradish peroxidase (HRP) as the secondary antibody. This is carried out by measuring the 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. Further, at least of the amino acid sequences constituting the present protein. An antibody having antigen-binding property to a continuous polypeptide consisting of usually 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 a reporter gene assay using the expression control region of this gene The present invention comprises the following steps (1) to (3), and is characterized by the ability to improve sugar / lipid metabolism Provide a method. 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 ability of the test substance to improve sugar / lipid metabolism 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 the gene encoding this protein (this gene)” is not particularly limited as long as it is a region capable of controlling the expression of OL16, but usually ranges from several kb to several tens of kb upstream of the chromosomal gene. Point to. For example, a region from the transcription start point to about 2 Kbp upstream, or a region consisting of a base sequence in which one or more bases are deleted, substituted or added in the base sequence of this region, and having the ability to control transcription of OL16 Can be mentioned. For example, (i) 5′-race method (5′-RACE method) (for example, it can be carried out using 5′full Race Core Kit (manufactured by Takara Bio Inc.)), oligo cap method, S1 primer mapping, etc. Step of determining the 5 ′ end by a usual method; (ii) 5′-upstream region is obtained using Genome Walker Kit (Clontech) etc., and promoter activity is measured for the obtained upstream region It can be identified by a method including steps;

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 regulatory region (transcriptional regulatory region) of the gene excised according to the technique can be incorporated on a plasmid containing a reporter gene operably linked to the region.
Reporter genes include β-glucuronidase (GUS), luciferase (LUC), chloramphenicol transacetylase (CAT), β-galactosidase, green fluorescent protein (GFP) and the like.
In addition, as long as the OL16 transcriptional regulatory region and the reporter gene operably linked to the region are introduced, the OL16 transcriptional regulatory function can be evaluated, that is, the expression level of the reporter gene can be quantitatively analyzed. There is no particular limitation. However, since a physiological transcriptional regulatory factor for OL16 is expressed and considered to be more appropriate for evaluation of the expression regulation of OL16, OL16-expressing cells are preferable as the introduced cells.
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 or protein expression inducer selected as described above is also a sugar / lipid metabolism improving agent, that is, a disease caused by impaired glucose tolerance, a therapeutic or preventive agent for a disease caused by obesity, or an obesity state It is a candidate substance for improving drugs.

II-4) Assay method using animals with abnormal sugar / lipid metabolism In other words, the following steps (1) to (3):
(1) a step of administering a test substance to an animal with abnormal sugar / lipid metabolism,
(2) measuring the expression level of OL16 or OL16 gene in an animal administered with the test substance, and comparing the expression level with a control animal not administered with the test substance; and
(3) Third step of evaluating the ability of the test substance to improve sugar / lipid metabolism using as an index that the expression level of OL16 or OL16 gene is larger than the expression level in the control animal based on the comparison result of the second step Have
Examples of animals used in the first step include mammals such as mice, rats, hamsters, guinea pigs, rabbits, dogs, monkeys and the like. “Animals with abnormal sugar / lipid metabolism” include animals loaded with a high-fat diet, model animals with diseases caused by impaired glucose tolerance (eg, dbdb mice, KKAy mice, Wister-fatty rats, Goto-Kakizaki (GK) Rat, Otsuka long-evens fatty acid (OLETF) rat, Akita mouse, pancreas-disrupted mouse and rat by administration of drugs such as streptozotocin, etc., obese model animals (for example, obob mouse, Ay mouse, Zucker-fatty rat, Otsuka long-evens fatty acid (OLETF) rats, mice induced by overeating by administration of drugs such as glutamic acid, rats and the like) and the like. An animal loaded with a high fat diet is an animal that has been administered a high fat diet. A high-fat diet is one in which the fat content is increased as compared to a normal diet for mice. Examples of the high-fat food that can be used in the screening method of the present invention include those in which the fat content of unit calories is increased from 4.8% to 30% by adding fat such as soybean oil and lard. It is done.
Administration of a test substance to an animal can be performed by a known method. For example, the administration method includes oral administration and parenteral administration (eg, intravenous injection, subcutaneous injection, intraperitoneal injection, local injection). The dose, administration interval, administration period, and the like can be appropriately set according to the test substance used, the type of animal, and the like. Examples of the test substance are the same as described above.
In the second step, the expression level of OL16 or OL16 gene in the animal to which the test substance is administered can be measured by the method described in II-1) or II-2) above. That is, it can be performed by collecting a biological sample from an animal and measuring the amount of a transcription product or translation product in the sample. As a biological sample to be collected from animals, among OL16-expressing tissues, those containing adipose tissue (eg, peripheral fat of the testicle, mesenteric fat, etc.) are desirable.
Subsequently, the expression level of OL16 in the animal administered with the test substance is compared with the expression level of OL16 in the control animal not administered with the test substance. The comparison of the expression level is preferably performed based on the presence or absence of a significant difference. The expression level of OL16 in the control animal not administered with the test substance may be the expression level measured in advance or the expression level measured simultaneously with respect to the measurement of the expression level of OL16 administered with the test substance. From the viewpoint of the accuracy and reproducibility of the experiment, the expression level measured simultaneously is preferable.

(III) Therapeutic agent or prophylactic agent The protein or fragment thereof, or the compound found by the method for searching for the present invention is used as it is or as a known pharmaceutically acceptable carrier (excipient, diluent). , Bulking agents, binders, lubricants, flow aids, disintegrants, surfactants, etc.) and conventional additives, etc., can be mixed to prepare a pharmaceutical composition. For example, excipients such as sucrose, starch, mannitol, sorbit, lactose, glucose, cellulose, talc, calcium phosphate or calcium carbonate, cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, Binders such as sucrose or starch, starch, carboxymethyl cellulose, hydroxypropyl starch, sodium-glycol starch, disintegrating agents such as sodium bicarbonate, calcium phosphate or calcium citrate, magnesium stearate, aerosil, talc or sodium lauryl sulfate Lubricants, citric acid, menthol, glycyrrhizin / ammonium salt, fragrance such as glycine or orange powder, sodium benzoate Preservatives such as um, sodium hydrogen sulfite, methyl paraben or propyl paraben, stabilizers such as citric acid, sodium citrate or acetic acid, suspensions such as methyl cellulose, polyvinyl pyrrolidone or aluminum stearate, dispersants such as surfactants, Examples include, but are not limited to, diluents such as water, physiological saline or orange juice, cocoa butter, base waxes such as polyethylene glycol or white kerosene.

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.
Formulations suitable for oral administration include a solution in which an effective amount of a substance is dissolved in a diluent such as water, saline, orange juice, a capsule containing an effective amount of the substance as a solid or granule, a sachet or Examples thereof include tablets, suspensions in which an effective amount of a substance is suspended in an appropriate dispersion medium, and emulsions in which a solution in which an effective amount of a substance is dissolved is dispersed in an appropriate dispersion medium and emulsified.
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. For example, a pharmaceutically acceptable carrier can be included.

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 and the like in vivo. 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.

(IV) Transgenic non-human animal The present invention further provides a transgenic non-human animal, or a tissue or cell derived therefrom.
The transgenic non-human animal of the present invention may be permanently present in a state in which the OL16 gene is introduced specifically into a fat cell and can be expressed in the fat cell. The species of the animal of the present invention is not particularly limited as long as it is a species capable of producing a transgenic animal. For example, non-human mammals such as mice, rats, hamsters, guinea pigs, rabbits, dogs, monkeys, etc. Can be mentioned. Preferred examples include rodent animals such as mice or rats.
The transgenic non-human animal of the present invention is useful as a model animal exhibiting an anti-obesity effect. In the model animal, a significant decrease in body weight and / or adipose tissue weight is observed in a high-fat diet as compared to a wild-type animal.
The transgenic non-human animal of the present invention can be used for analyzing pathological conditions such as diseases associated with abnormal glycolipid metabolic functions. Here, pathological analysis means analyzing all phenomena in the living body caused by a disease accompanied by abnormal glycolipid metabolism.
That is, the transgenic non-human animals of the present invention and wild-type non-human animals bred under standard dietary conditions or dietary obesity-inducing conditions (dietary load test) are compared in the period from embryonic period to lethality. , Development, observation of living behavior, histopathological examination or biochemical examination can be carried out. For observation and examination in these pathological conditions analysis, methods commonly used by those skilled in the art may be used, and reference can be made to “Transgenic Animals” Kenichi Yamamura et al. (Kyoritsu Shuppan Co., Ltd.).
For example, the total RNA extracted from the tissue of the transgenic non-human animal of the present invention is analyzed by a method such as PCR real-time quantitative analysis or Northern blot analysis, and compared with the value in a wild-type non-human animal to express in the tissue. Can be identified. From the results, it is possible to search for a marker gene whose expression fluctuates in a disease associated with abnormal glycolipid metabolic function, and thus can be used as an index for elucidating a causal factor of the disease.
The animals of the present invention can be produced by known methods or the methods described in the Examples herein. More particularly, the animal of the present invention provides adipocyte-specific expression, eg, in an animal's fertilized egg or other cells (eg, unfertilized egg, sperm or their precursor cells) at an early stage of development. It can be produced by introducing an OL16 gene (the above-mentioned OL16 expression vector) operably linked to a promoter to be enabled. Examples of gene introduction methods include electroporation, lipofection, aggregation, calcium phosphate coprecipitation, and microinjection. The animal of the present invention may also be an animal produced by crossing an OL16 transgenic animal produced in this way with another animal of the same species (eg, the above-mentioned model animal with abnormal sugar / lipid metabolism).
The tissue or cell derived from the animal of the present invention is not particularly limited as long as it is any tissue or cell that can be collected from the animal of the present invention. More specifically, examples of the tissue or cells derived from the animal of the present invention include adipose tissue or adipocytes, bone marrow stromal cells, fibroblasts, blood mononuclear cells, but adipose tissue or adipocytes. Is preferred.
The animal of the present invention or a tissue or cell derived therefrom is, for example, the screening method of the present invention, a marker gene for a disease or condition for which improvement in sugar / lipid metabolism ability is desired (that is, a marker whose expression varies when the disease condition is improved). Screening, or adipocyte marker gene screening, analysis of the pathological mechanism of a disease or condition in which improvement of sugar / lipid metabolism ability is desired, analysis of the pathological mechanism of a disease or condition caused by obesity, and the like. These measure, for example, the expression profile (particularly the expression profile of adipocytes) in the animal of the present invention using a microarray, a protein chip (eg, a non-antibody chip such as an antibody chip or a chip made by Cyphergen), etc. It can be done by expression profile analysis, including comparing to the expression profile of a control animal.
The contents of all publications, including patents and patent application specifications cited in this specification, are hereby incorporated by reference herein to the same extent as if all were explicitly stated. Is.
The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.

Example 1: Production of adipocyte -specific OL16 transgenic mice (aP2-OL16 Tg mice) In order to investigate the role of OL16 in obesity pathology using mice in which OL16 is overexpressed in adipocytes, aP2 (fatty acid binding protein ) Promoter 5.4 Kbp (see Ross et al., Proceeding of the national academy of scince of United States of America Vol. 87: 9590-9594 (1990)) aP2-OL16 trans that specifically expresses OL16 in adipocytes A transgenic (aP2-OL16 Tg) mouse was created. Specifically, aP2-OL16 cDNA solution was injected into a C57BL / 6 fertilized egg and then returned to the foster mother's uterus to obtain a pup mouse. As a result of screening, it was found that 3 mice had aP2-OL16 DNA. Furthermore, it became clear that these mice could transmit aP2-OL16 to the next generation, and three strains of aP2-OL16-introduced mice could be established.
Next, it was confirmed whether or not the obtained mice expressed OL16 specifically in adipocytes. As a result, expression in adipocytes was confirmed.

Example 2: Analysis of the phenotype of aP2-OL16Tg mice fed a high fat diet
2-1 Changes in body weight and tissue weight of aP2-OL16Tg mice under high-fat diet The aP2-OL16Tg mice (line 22) under high-fat diet showed a marked suppression of weight gain compared to wild-type mice ( FIG. 1). In addition, aP2-OL16Tg mice under high-fat diets were more prominent in inhibiting the increase in the circumference of the epididymis, mesentery, and retroperitoneal fat compared to wild-type mice (FIG. 2).

2-2 Glucose tolerance test in aP2-OL16Tg mice under high fat diet (2g / Kg intraperitoneally administered)
In aP2-OL16Tg mice (line 22 27 weeks old) under the high-fat diet, a decrease in blood glucose level after loading was observed and an improvement in glucose metabolism was observed (FIG. 3).

  The agent for improving sugar / lipid metabolic ability of the present invention is useful as a therapeutic or preventive agent for diseases caused by impaired glucose tolerance or obesity, or as an agent for improving obesity. In addition, by the assay method of the present invention, a sugar / lipid metabolic ability improving agent by a new mechanism can be evaluated.

It is a figure which shows the correlation with the blood level of OL16 in human and body weight. In the figure, ACAM represents OL16. It is a figure which shows the weight change of the aP2-OL16Tg mouse | mouth under a high fat diet. In the figure, ACAM represents OL16. It is a figure which shows the tissue weight of the aP2-OL16Tg mouse (age 27 weeks a week) under a high fat diet. In the figure, ACAM represents OL16. It is a figure which shows the change of the blood glucose level after the glucose tolerance test (intraperitoneal, 2 g / Kg) in the aP2-OL16Tg mouse (week 27 weeks old) under a high fat diet. In the figure, ACAM represents OL16.

Claims (19)

  An agent for improving sugar / lipid metabolism, comprising OL16 or a substance that promotes expression of OL16 as an active ingredient.   The agent for improving sugar / lipid metabolic ability according to claim 1, wherein the substance that promotes the expression of OL16 is a gene encoding OL16.   The agent for improving sugar / lipid metabolic ability according to claim 1, wherein the substance that promotes the expression of OL16 is an expression vector containing a gene encoding OL16.   The agent for improving sugar / lipid metabolic ability according to any one of claims 1 to 3, which is used as a preventive and therapeutic agent for a disease caused by impaired glucose tolerance or a disease caused by obesity.   The agent for improving sugar / lipid metabolic ability according to any one of claims 1 to 3, which is used as an agent for improving obesity.   A test method for the ability of a test substance to metabolize sugar / lipid, using as an index whether or not the test substance promotes the expression of OL16 or a gene encoded by OL16. The assay method according to claim 6, 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 OL16;
(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 ability of the test substance to metabolize sugar / lipid 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 assay method according to claim 6, comprising the following steps (1) to (3):
(1) a first step of contacting a test substance with a cell capable of expressing OL16;
(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 ability of the test substance to metabolize sugar and lipid based on the comparison result of the second step, using as an index whether or not the test substance increases the expression level of the protein. The assay method according to claim 6, 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 that is operably linked to an expression control region of a gene encoding OL16;
(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 sugar / lipid metabolic ability 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 claim 6, comprising the following steps (1) to (3):
(1) A step of administering a test substance to an animal with abnormal sugar / lipid metabolism,
(2) a step of measuring the expression level of OL16 or OL16 gene in an animal administered with the test substance and comparing it with the expression level in a control animal not administered with the test substance;
(3) A third step of evaluating the sugar / lipid metabolic ability of the test substance using as an index that the expression level of OL16 or OL16 gene is larger than the expression level of the control animal based on the comparison result of the second step.   The assay method according to claim 10, wherein the animal with abnormal sugar / lipid metabolism is a high-fat diet-fed animal.   The assay method according to claim 10 or 11, wherein the animal with abnormal sugar / lipid metabolism is a mouse or a rat.   A saccharide, wherein the sugar / lipid metabolic ability improving agent is selected by using the sugar / lipid metabolic ability of the test substance measured by the assay method according to claim 6 as an index. -A method for searching for an agent for improving lipid metabolism ability.   An agent for improving the ability to metabolize sugar and lipid, comprising as an active ingredient a substance selected by the searching method according to claim 13 or a pharmaceutically acceptable salt thereof.   The agent for improving sugar / lipid metabolic ability according to claim 14, which is used as a preventive and therapeutic agent for a disease caused by impaired glucose tolerance or a disease caused by obesity.   The agent for improving sugar / lipid metabolic ability according to claim 14, which is used as an agent for improving obesity.     A transgenic non-human animal into which the OL16 gene has been introduced, characterized in that OL16 is expressed specifically in adipose tissue.   The transgenic non-human animal according to claim 17, which is an anti-obesity or anti-diabetic model animal.   Adipose tissue or adipocytes derived from the transgenic non-human animal according to claim 17.

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