WO2004040301A1 - Method of diagnosing type 2 diabetes - Google Patents

Abstract

It is intended to provide a method of diagnosing type 2 diabetes whereby type 2 diabetes can be diagnosed by analyzing the expression of a gene in a tissue which can be easily sampled (in particular, the onset risk of type 2 diabetes in future can be presumed before the onset of type 2 diabetes, thereby enabling early detection of type 2 diabetes). This method of diagnosing type 2 diabetes is characterized by diagnosing type 2 diabetes with the use of, as an indication, the expression level of CAPN10 gene or IRS-1 gene in leukocytes collected from the blood of a subject.

Description

 How to diagnose type 2 diabetes

Technical field

 The present invention relates to a method for diagnosing type 2 diabetes and a diagnostic kit, a method for screening a substance having a preventive / therapeutic effect for type 2 diabetes, and a screenin.

Related to kits for book

 Background art

 The number of people with diabetes is steadily increasing on a global scale, and is expected to exceed 200 million in FY2010. Diabetes is broadly classified into type 1 and type 2 based on its etiology, but most of the diabetic patients are occupied by type 2 diabetic patients, and overcoming it is one of the major challenges for humankind.

 Type 1 diabetes is a disease in which the islets of Langerhans are inflamed and the ability of jS cells to secrete insulin is significantly reduced, and the disease is an insulin-dependent disease that cannot survive without supplementing insulin.

In type 2 diabetes, insufficient action of insulin appears due to other causes, resulting in hyperglycemia.Environmental factors such as obesity, overeating, lack of exercise, stress, etc. are greatly involved, and relatively elderly people after middle age It is easy to develop in obese people. Type 2 diabetes generally presents a non-insulin-dependent pathology, with diet and exercise therapies being the basis of treatment. Drug therapy is the next stage of diet and exercise therapy, and insulin therapy is used if treatment is still difficult. Symptoms such as polydipsia, polyuria and nocturnal urine are seen at the onset of diabetes, but few patients are aware of these initial symptoms, and many patients are associated with complications Diabetes is imperceptible because symptoms cannot be realized until symptoms appear, and when it is discovered, complications appear and treatment is often extremely difficult. Therefore, early detection and early treatment are extremely important for overcoming diabetes.However, since the pathogenesis of type 2 diabetes is still unclear compared to type 1 diabetes, early detection and early treatment are difficult. May be. Therefore, various genetic approaches have been taken to elucidate the pathogenesis of type 2 diabetes. Recently, SNP s analysis in diabetic patients ゃ Inherited nucleotide sequence abnormality has been found in genes by haplotype analysis Is becoming clearer. For example, a change in the incidence of diabetes due to an abnormality in the nucleotide sequence (Altshuler, D. et al., At Genet, 2000. 26 (1) p. 76-80; Yen, CJ et al., Biochem Biophys Res Commun, 1997. 241 ( 2) p.270-4) Decreased function of J6 kidney j6 cells (Maechler, P. and CB Wollheim, Nature, 2001. 4 14 (6865) p.807-12; Bell, GI and KS Polonsky, Nature, 2001. 414 (6865) p. 788-91), and also changes in drug sensitivity (Umekawa, T. et al., Diabetes, 1999. 48 (1) p. 117-20; Hof fs tedt, J. et al., Diabetes , 1999. 48 (1) p.203-5). However, the genes responsible for type 2 diabetes have not been completely identified, and much remains to be known about the pathogenesis of type 2 diabetes.

 Other genetic approaches include gene expression analysis. Gene expression analysis analyzes the expression state (phenotype) of a gene, and is essentially different from SNP s analysis, etc., which analyzes congenital abnormalities of a gene (genotype). It is also advantageous in that the current situation of patients taking into account environmental factors can be ascertained.

However, gene expression analysis so far has been performed to examine changes in the expression of genes related to glucose metabolism and lipid metabolism in major target organs of insulin, such as liver, skeletal muscle, fat, and kidney. This is clinical It is difficult to apply it to diagnose type 2 diabetes. In other words, when targeting a major target organ of insulin, it is difficult to sample the sample, so it is difficult to diagnose type 2 diabetes based on gene expression analysis. Disclosure of the invention

 Thus, the present invention firstly makes it possible to diagnose type 2 diabetes by analyzing gene expression in tissues that are easily sampled (especially before type 2 diabetes onset, is it possible to develop type 2 diabetes in the future? It is possible to provide a diagnostic method and a diagnostic kit for type 2 diabetes.

 Also, the present invention provides, secondly, a substance having a type 2 diabetes preventive / therapeutic effect, which can screen a substance having a type 2 diabetes preventive / therapeutic effect by analyzing gene expression in a tissue which is easily sampled. An object of the present invention is to provide a screening method and a screening kit.

 In order to achieve the above object, the present invention provides the following method for diagnosing type 2 diabetes and a diagnostic kit, and a method for screening a substance having a preventive / therapeutic effect for type 2 diabetes and a kit for screening.

 (1) A method for diagnosing type 2 diabetes, which comprises diagnosing type 2 diabetes using the expression level of CAPN10 gene or IRS-1 gene in leukocytes collected from the blood of the subject as an index.

 (2) The diagnostic method according to (1), wherein the expression level is measured based on the abundance of mRNA encoding CAPN10 or IRS-1.

(3) The expression level is determined based on the abundance of CAP N 10 or IRS-1. The diagnostic method according to the above (1), wherein the measurement is carried out.

 (4) When the expression level of CAPN10 gene or IRS-1 gene in leukocytes of the subject is lower than the expression level of CAPN10 gene or IRS-1 gene in leukocytes of healthy subjects Diagnosing that the subject is likely to develop type 2 diabetes in the future or that the subject is currently likely to develop type 2 diabetes

(1) The diagnostic method described.

 (5) A kit for diagnosing type 2 diabetes, comprising an oligonucleotide or a polynucleotide capable of hybridizing to a nucleic acid encoding CAPN10 or IRS-1.

 (6) A kit for diagnosing type 2 diabetes, comprising an antibody capable of reacting with CAPN10 or IRS-1 or a fragment thereof.

 (7) After administering the candidate substance to a type 2 diabetes model animal, using the index of the effect of improving the expression level of the CAPN10 gene or IRS-1 gene in leukocytes collected from the blood of the animal as an index, the type 2 of the candidate substance A method for screening a substance having a preventive and therapeutic effect on type 2 diabetes, which comprises determining the preventive and therapeutic effects of diabetes.

 (8) A kit for screening a substance having a type 2 diabetes preventive / therapeutic effect, comprising an oligonucleotide or a polynucleotide capable of hybridizing to a nucleic acid encoding CAPN10 or IRS-1.

 (9) A kit for screening a substance having a preventive / therapeutic effect for type 2 diabetes, comprising an antibody or a fragment thereof that can react with CAPN10 or IRS-1. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows changes in body weight of OLETF rats and LETO rats. is there.

 FIG. 2 is a graph showing changes in glucose tolerance of OLETF rats and LET0 rats.

 Fig. 3 shows IR (Fig. 3A), SHIP2 (Fig. 3B), PPARa (Fig. 3C), CAPN10 (Fig. 3D) and IRS-1 in leukocytes of OLETF rats and LETO rats. FIG. 3E shows the results of mRNA expression analysis of (FIG. 3E), in which white (mouth) indicates the results of LETO rats, and black (騸) indicates the results of OLE.

The results for TF rats are shown. BEST MODE FOR CARRYING OUT THE INVENTION

 The method for diagnosing type 2 diabetes of the present invention is characterized in that diagnosis of type 2 diabetes is performed using the expression level of the CAPN10 gene or the IRS-1 gene in leukocytes collected from the blood of the subject as an index.

 The method for collecting white blood cells from the blood of the subject is not particularly limited. For example, white blood cells can be collected by selectively dissolving red blood cells in blood and then centrifuging. Leukocytes include neutrophils, eosinophils, basophils, lymphocytes and monocytes, and one or more of these may be used as a specimen. It may be a mixture of

The CAPN10 gene is a gene encoding CAPN10 (CalpainlO, calpain10). CAPN10 is a cysteine protease that is non-specifically expressed. CAPN10 does not have the calcium-binding domain that calpain normally has, but instead has a T domain (Horikawa, Y. et al., Nat Genet, 2000. 26 (2) p .163-7 5). The function of CAPN 10 is, for example, involved in the degradation of IRS-1 (Smith, LK et al., Biochem Biophys Res Commun, 1993. 196 (2) p. 767-72), incorporated into the A-kinase system and involved in adipocyte differentiation (Patel, YM and MD Lane, Proc Natl Acad Sci USA, 1999.96 (4) p.1279-84), protein Hydrolysis of kinase C (Suzuki, K. et al., FEBS Lett, 1987. 220 (2) ρ · 271-7) and the like are known.

 The IRS-1 gene is a gene that encodes IRS-1 (I lin receptor substrate-1, insulin receptor substrate-1). IRS-1 is a protein involved in the mechanism of insulin action. In other words, when insulin binds to the insulin receptor on the cell surface, various proteins, including IRS-1 bound to the insulin receptor, are activated one after another, and their signals are transmitted. GLUT4) activates and transports glucose from outside to inside the cell.

 The nucleotide sequence of the CAPN10 gene may differ among subjects due to polymorphisms, isoforms, etc. Contained in the CAPN10 gene. The same applies to the IRS-1 gene.

 “Expression level of CAPN10 gene” This includes the level of transcription of the 1 ^ 10 gene into 111 RNA and the level of translation into protein. Similarly, the “expression level of the IRS_1 gene” includes the level of transcription of the IRS-1 gene into mRNA and the level of translation into protein. Therefore, the expression level of the CAPN10 gene or IRS-1 gene is determined by the amount of mRNA that encodes CAPN10 or IRS-1 in the sample, or the CAPN10 or IRS-1 gene in the sample. It can be measured based on the abundance of IRS-1.

In measuring the abundance of mRNA encoding CAPN10 or IRS-1, known gene analysis techniques, for example, hybridization Technology (eg, Northern hybridization method, dot blot method)

, DNA microarray method, etc.) and gene amplification technology (eg, RT-PCR etc.) can be used.

 When using the hybridization technique, an oligonucleotide or polynucleotide capable of hybridizing to the nucleic acid encoding CAPN10 or IRS-1 can be used as a probe, and when using the gene amplification technique. Can use the oligonucleotide as a primer.

 Nucleic acids encoding CAPN10 or IRS-1 include both DNA and RNA, including, for example, mRNA, cDNA, cRNA and the like. The nucleotides constituting the oligonucleotide or the polynucleotide may be any of deoxyliponucleotides and liponucleotides, or unnatural nucleotides. The base length of the oligonucleotide is usually 15 to 100 bases, preferably 18 to 40 bases, and the base length of the polynucleotide is usually 200 to 300 bases, preferably 500 to 100 bases. 100 bases.

The nucleotide sequence of an oligonucleotide or polynucleotide that can hybridize to a nucleic acid encoding CAPN 10 or IRS-1 is designed based on the nucleotide sequence of CAPN 10 gene or IRS-1 gene. be able to. For example, a region containing CDS is selected from cDNAs encoding CAPN 10 or IRS-1, and the nucleotide sequence of the oligonucleotide or polynucleotide is designed so as to hybridize to the region. In addition, it is possible to hybridize to a region at the 5 'end or 3' end of the CDS region, or to hybridize to a region extending from the CDS region to a region at the 5 'end or 3' end thereof. Can be designed. For the designed oligonucleotide or polynucleotide, It is preferable to use it as a probe or a probe to confirm whether or not it hybridizes to the target nucleic acid. The nucleotide sequence of cDNA encoding human CAPN10 is shown in SEQ ID NO: 1, and the nucleotide sequence of cDNA encoding human IRS-1 is shown in SEQ ID NO: 2. In the cDNA described in SEQ ID NO: 1, the region consisting of the 24th to 24th nucleotides is the CDS region, and in the cDNA shown in SEQ ID NO: 2 it is comprised of the 1021 to 4749th nucleotides The area is the CDS area. When an oligonucleotide or a polynucleotide is used as a primer, a restriction enzyme recognition sequence, a tag, or the like can be added to the 5 'end. When used as a probe, a label such as a fluorescent dye or a radioisotope can be added.

 A specific method for measuring the abundance of mRNA that encodes CAPN 10 or IRS-1 will be described using RT_PCR as an example. After extracting total RNA from leukocytes collected from the blood of the subject and synthesizing cDNA from the extracted total RNA, the synthesized cDNA is type II, and cDNA encoding CAP N10 or IRS-1 PCR is performed using a primer set capable of hybridizing to the above, and the amount of mRNA encoding CAPN10 or IRS-1 can be measured by quantifying the PCR-amplified fragment. At this time, the PCR is performed under conditions such that the amount of PCR amplified fragment production reflects the amount of the initial type III cDNA (for example, the number of PCR cycles at which the PCR amplified fragment increases exponentially).

 The method for quantifying the PCR-amplified fragment is not particularly limited. For quantification of the PCR-amplified fragment, for example, a quantification method using a radioisotope (RI), a quantification method using a fluorescent dye, or the like can be used. it can.

Examples of the quantification method using RI include: (i) adding a RI-labeled nucleotide (for example, ³² P-labeled dCTP, etc.) as a substrate to a reaction solution, incorporating the nucleotide into an amplified PCR fragment, and PCR amplification. Fragments; RI-labeled and P Separating the CR amplified fragment by electrophoresis, etc., measuring the radioactivity to quantify the PCR amplified fragment, (ii) RI-labeling the PCR amplified fragment by using RI-labeled primers, Separating the amplified CR fragments by electrophoresis, etc. and measuring the radioactivity to quantify the amplified PCR fragments. (Iii) Electrophoresing the amplified PCR fragments, blotting them onto a membrane, and performing RI labeling. A method in which the probe is hybridized, the radioactivity is measured, and the PCR amplified fragment is quantified. The radioactivity can be measured using, for example, a liquid scintillation counter, an X-ray film, an imaging plate, or the like.

 Quantitative methods using fluorescent dyes include (i) using a fluorescent dye that interacts with double-stranded DNA (eg, ethidium bromide (EtBr), SYBR GreenI, PicoGreen, etc.) A method for quantifying PCR amplified fragments by staining and measuring the intensity of fluorescence emitted by excitation light irradiation, and (ii) labeling PCR amplified fragments with a fluorescent dye by using a fluorescent dye-labeled primer. After the PCR amplified fragment is separated by electrophoresis or the like, the fluorescence intensity is measured to quantify the PCR amplified fragment. The fluorescence intensity can be measured using, for example, a CCD camera, a fluorescence scanner, a spectrofluorometer, or the like.

 When RT-PCR is used, the gene amplification process is monitored in real time using a commercially available device such as ABI PRISM 7700 (Applied Biosystems), so that the PCR-amplified fragment can be quantitatively determined. Analysis can be performed. ·

The measured value of the expression level of the CAPN10 gene or the IRS-1 gene is based on the measured value of the expression level of a gene whose expression level does not fluctuate greatly (for example, the housekeeping gene such as the / 3-actin gene and the GAPDH gene). It is preferable to make correction. When measuring the abundance of CAPN10 or IRS-1, known protein analysis techniques, for example, western blotting using an antibody or a fragment thereof capable of reacting with CAPN10 or IRS_1, Sedimentation, ELISA and the like can be used.

Antibodies capable of reacting with CAPN 10 or IRS-1 include both monoclonal antibodies and polyclonal antibodies, and “fragments thereof” include those that can react with CAPN 10 or IRS-1. , Any fragments. Examples of antibody fragments include Fab fragments, F (ab) ' ₂ fragments, and single-chain antibodies (scFv). The term "capable of reacting with CAPN10 or IRS-1" includes the case where it reacts with any part of CAPN10 or IRS-1. It is preferable that the antibody or a fragment thereof that can react with CAPN10 or IRS-1 reacts with CAPN10 or IRS-1, but does not react with other proteins contained in leukocytes.

 An antibody capable of reacting with CAPN10 or IRS-1 can be obtained, for example, as follows.

 As the immunizing antigen, some or all of CAPN 10 or IRS-1 can be used. Examples of the antigen for immunization include: (i) a crushed cell or tissue expressing CAPN10 or IRS-1 or a purified product thereof;

(ii) cDNA encoding CAPN 10 or IRS-1 (for example, the nucleotide sequence of SEQ ID NO: 1 for CAPN 10 and SEQ ID NO: 2 for IRS-1) using DNA recombination technology A recombinant protein expressed by introducing cDNA into a host such as Escherichia coli, insect cells or animal cells,

(iii) Chemically synthesized peptides and the like can be used.

In the preparation of polyclonal antibodies, mammals such as rats, mice, guinea pigs, rabbits, sheep, horses, horses, and horses are first immunized with an antigen for immunization. During immunization, complete Freund's to induce antibody production It is preferable to immunize a plurality of times after emulsifying with an adjuvant (FCA), Freund's incomplete adjuvant (FIA), or another immunological adjuvant. The antibody titer against the insulin signaling control protein is measured, and blood is collected after the antibody titer has increased to obtain an antiserum.

 In preparing a monoclonal antibody, a mammal is immunized with an immunizing antigen in the same manner as in the case of the polyclonal antibody, and then the antibody-producing cells are collected. Examples of the antibody-producing cells include spleen cells, lymph node cells, thymocytes, peripheral blood cells and the like, and spleen cells are generally used. Next, in order to obtain a hybridoma, cell fusion between the antibody-producing cell and the myeloid cell is performed. After the cell fusion treatment, the cells are cultured using a selection medium to select the desired hybridoma. Next, the presence or absence of the target antibody is screened in the culture supernatant of the grown hybridoma. Next, hybridomas are cloned by a limiting dilution method, a soft agar method, a fibrin gel method, a fluorescence excitation cell sorter method, etc., and finally, a hybridoma producing a monoclonal antibody is obtained. As a method for collecting a monoclonal antibody from the obtained hybridoma, an ordinary cell culture method or the like can be used. In addition, after transplanting the hybridoma into the abdominal cavity of a mouse or the like, ascites may be collected, and a monoclonal antibody may be obtained from the ascites.

 If purification of polyclonal or monoclonal antibodies is required, select an appropriate method such as salting out with ammonium sulfate, gel chromatography, ion exchange chromatography, affinity chromatography, etc. These can be used in combination.

When quantifying the expression level of CAPN 10 or IRS-1 using an antibody or a fragment thereof capable of reacting with CAPN 10 or IRS-1, for example, radioimmunoassay (RIA), enzyme immunization Assay (E IA), Chemiluminescence Assay (CL IA) And fluorescence immunoassay (FIA) can be used. Specifically, after capturing CAP N10 or IRS-1 in a sample using a solid phase carrier (eg, immunoplate, latex particles, etc.) to which the antibody has been bound by physical adsorption or chemical binding, etc. An antibody in which the captured CAPN 10 or IRS-1 is immobilized on a solid support is a labeled antibody having a different antigen recognition site for CAPN 10 or IRS-1 (for example, peroxidase, alkaline An enzyme labeled with an enzyme such as phosphatase or a fluorescent substance such as phloretsense or umberiferon).

 The measurement of the abundance of CAPN 10 or IRS-1 can also be performed by measuring the activity of CAPN10 or IRS-1. The activity of CAPN10 or IRS-1 can be measured, for example, by a known method such as a Western plotting method or an ELISA method using an antibody or a fragment thereof capable of reacting with CAPN10 or IRS_1. it can.

 Because type 2 diabetes is a slowly progressing disease, type 2 diabetes progresses slowly even before obvious symptoms of type 2 diabetes such as hyperglycemia and diabetes appear (that is, before the onset of type 2 diabetes). You may have.

The method for diagnosing type 2 diabetes of the present invention utilizes the fact that the expression level of the CAPN10 gene or IRS-1 gene in leukocytes changes differently from the normal expression level as the type 2 diabetes progresses. Diagnosis of type 2 diabetes is performed using the expression level of the CAPN10 gene or IRS-1 gene in leukocytes collected from the blood of the subject as an index. That is, since the expression level of the CAP N10 gene or IRS-11 gene in leukocytes is lower than the normal expression level with the progression of type 2 diabetes, the CAP N10 gene or IRS 11 gene or IRS When the expression level of the RS-1 gene is lower than the expression level of the CAPN10 gene or the IRS-1 gene in leukocytes of a healthy subject, the subject will develop type 2 diabetes in the future May be diagnosed as being likely to have or have presently developed type 2 diabetes. In the method for diagnosing type 2 diabetes according to the present invention, the tissue that needs to be sampled from the subject is blood, and blood is easier to sample than other tissues. Can be.

 When comparing the expression levels of CAPN10 gene or IRS-1 gene in leukocytes between a subject and a healthy subject, the expression levels of a plurality of healthy subjects (healthy subjects) are measured and the It is preferable to set a normal range from the distribution and determine whether the expression level of the subject is higher than or lower than the normal range.

 The healthy subject group can be constituted by arbitrarily selecting a plurality of healthy subjects, but is preferably constituted by healthy subjects of the same age or generation as the subject. This is to eliminate as much as possible the effect of the age difference on the expression level of the CAPN10 gene or IRS-1 gene.

 The kit for diagnosing type 2 diabetes of the present invention comprises CAPN10 or IRS-1 as a reagent for measuring the expression level of the CAPN10 gene or IRS-1 gene in leukocytes collected from the blood of a subject. It is characterized by containing an oligonucleotide or polynucleotide capable of hybridizing to the encoding nucleic acid, or an antibody or a fragment thereof capable of reacting with CAPN10 or IRS-1.

With the use of the kit for diagnosing type 2 diabetes of the present invention, diagnosis of type 2 diabetes can be performed by measuring the expression level of the CAPN10 gene or the IRS-1 gene in leukocytes collected from the blood of the subject. Can be. The kit for diagnosing type 2 diabetes of the present invention may be in any form as long as it contains the above-mentioned oligonucleotide or polynucleotide, or the above-mentioned antibody or fragment thereof, and may contain any reagent, instrument, etc. it can. Type 2 diabetes diagnostic kit of the present invention, when containing the oligonucleotide or polynucleotide may, P reagents required CR (e.g. H ₂ 0, buffer, Mg C l _2, dNTPs mix, T a Q polymerase And one or more of reagents necessary for quantification of PCR amplified fragments (eg, RI, fluorescent dye, etc.), DNA microarrays, DNA chips and the like. When the kit for diagnosing type 2 diabetes of the present invention contains the above-mentioned antibody or a fragment thereof, a solid phase carrier (eg, immunoplate, latex particles, etc.) for immobilizing the antibody or the fragment thereof may be used. Labeling (eg, enzymes, fluorescent substances, etc.), various reagents (eg, enzyme substrates, buffers, diluents, etc.) Types or two or more types can be included.

 The method for screening a substance having a preventive / therapeutic effect of type 2 diabetes according to the present invention comprises administering a candidate substance to a type 2 diabetes model animal, and then CAPN10 gene or IRS_1 in leukocytes collected from the blood of the animal. The preventive and therapeutic effects of the candidate substance on type 2 diabetes are determined using the effect of improving the expression level of the gene as an index.

 Since the expression level of the CAP N10 gene or IRS_1 gene in leukocytes decreases from the normal expression level with the progression of type 2 diabetes, the CAP N 10 gene or IRS_1 gene in the leukocytes of a type 2 diabetes model animal is By selecting a substance having an effect of improving the expression level of the IRS-11 gene, a substance having a preventive / therapeutic effect for type 2 diabetes can be screened.

In the screening method of the present invention, the tissue that needs to be sampled from a type 2 diabetes model animal is blood, and blood is easier to sample than other tissues. Can be easily determined. The “effect of improving the expression level of the CAPN 10 or IRS-1 gene” includes both the effect of returning the expression level of the CAPN 10 or IRS-1 gene to the normal expression level and the effect of approaching the normal expression level. And the effects exerted on any steps such as transcription and translation of the CAP N10 or IRS-1 gene, and expression of the activity of CAPN10 or IRS-1. In the screening method of the present invention, as a type 2 diabetes model animal, an animal in which the expression level of the CAPN10 gene or IRS_1 gene in leukocytes is lower than the normal expression level (for example, rat, mouse, etc.) , Guinea pigs, egrets). Such animals are those that are likely to develop type 2 diabetes in the future or that are currently developing diabetes.

 As a model animal to which the candidate substance is administered, a transgenic animal in which the expression level of the CAPN10 gene or the IRS-1 gene has been artificially reduced can also be used. The decrease in the expression level of the CAP N10 gene or the IRS-1 gene in the transgenic animal includes a state in which the expression level of the CAPN10 gene or the IRS_1 gene is reduced, and the state of the CAPN10 or IRS-1 gene. Includes any state in which the degradation of _1 is enhanced. '

The screening method of the present invention utilizes the fact that a change in the expression level of the CAPN10 gene or IRS-1 gene in leukocytes is an indicator of the possibility of the onset of type 2 diabetes in the future or present, After administering a candidate substance to a type 2 diabetes model animal, the effect of improving the expression level of the CAP N10 gene or IRS-1 gene in leukocytes collected from the blood of the animal is used as an index to evaluate the preventive and therapeutic effects of the candidate substance on diabetes. Make a decision. That is, the candidate substance is used as an index based on whether the expression level after administration of the candidate substance has returned to or approached the normal expression level. The effect of preventing and treating type 2 diabetes is determined, and based on the results, substances having the effect of preventing and treating diabetes are screened. The normal expression level is preferably determined by measuring the expression levels of a plurality of healthy animals and determining the distribution of the values.

 The screening kit for a substance having a cancer preventive / therapeutic effect of the present invention encodes CAPN10 or IRS-1 as a reagent for measuring the expression level of CAPN10 gene or IRS-1 gene in a sample. Or an oligonucleotide capable of hybridizing to a nucleic acid, or an antibody capable of reacting with CAPN10 or IRS-1, or a fragment thereof.

 The screening kit of the present invention can be used to measure the expression level of the CAPN10 gene or IRS-1 gene in leukocytes collected from the blood of a subject, thereby screening for a substance having a cancer prevention / treatment effect. You can do one-two.

 The screening kit of the present invention may be in any form as long as it contains the above-mentioned oligonucleotide or polynucleotide, or the above-mentioned antibody or a fragment thereof, and is exemplified in the type 2 diabetes diagnosis kit of the present invention. In addition to various reagents and instruments, model animals and the like can be included.

〔Example〕

 Hereinafter, the present invention will be described specifically with reference to Examples.

 1.0 Diabetes onset in LETF rats

 1.1 Experimental materials and methods

 1.1.1 Animals used

Male 0LETF (0tsuka-Long-Evans-Tokushima-Fatty) rats and LET0 (Lo) were maintained as strains at the Tokushima Research Laboratory, Otsuka Pharmaceutical Co., Ltd. ng-Evans-Tokushima-Otsuka) rats were used. OLETF rats are spontaneous diabetes mellitus model animals showing symptoms similar to type 2 diabetes (non-insulin-dependent diabetes) (Kawano, K. et al., Diabetes Res Clin Pract, 1994. 24 Suppl p. S317-20 ), LETO rats are control animals that do not develop diabetes at all. As described above, there is a merit that a gene expression state before and after the onset of diabetes can be analyzed by using a model animal that definitely develops diabetes and a model animal that does not develop diabetes at all. 0LETF rats and LET0 rats were 4 weeks old and were provided by Otsuka Pharmaceutical Co., Ltd. Tokushima Laboratory.

 Rats are animals in a light-dark environment with artificial lighting (light period 7: 00-21: 00, dark period 21: 0 0-7: 00), constant temperature (2rC ± 0.5 ° C), relative humidity (58%) They were bred in the laboratory and had free access to chow and water.

1.1.2 Experimental procedure

 In 0LETF rats and LET0 rats (n = 5-6), body weight was measured from 5 weeks of age to the end of the experiment (24 weeks of age). A glucose tolerance test was performed the week before sampling.

1.1.3 Glucose Tolerance Test (GTT)

 The glucose tolerance test (hereinafter referred to as “GTT”) used the method of Hotta et al. (Hotta, K. et al., Biochem Biophys Acta, 1996. 1289 (1) p.145-9).

That is, at 5 weeks and 23 weeks of age, rats were fasted for 12 hours from 21:00 and 1 g / kg BW glucose solution was examined at 5 weeks and 23 weeks of age to observe changes in glucose tolerance in OLETF rats and LETO rats. Was administered intraperitoneally using a 27G syringe. One hour later, blood was collected from the tail vein and measured for blood glucose. 1.1. Blood glucose measurement method

 Blood glucose was measured by the glucose oxidase method using the glucose B test method (Trinder, P., J Clin Pathol, 1969. 22 (2) p. 158-61).

 That is, the blood sampled from the tail vein was allowed to stand on ice for 15 to 20 minutes, and then centrifuged at 100 ° C for 2 minutes at 4 ° C. After diluting 2 μl of the centrifuged supernatant with physiological saline, 20 × L was mixed with 1 mL of a color test solution of glucose B test protein. After incubating at 37 ° C for 1 hour, the absorbance at 505 nm was measured.

1.1.5 Reagents used

 Glucose B Test Co. was purchased from Wako Pure Chemical Industries, Ltd. Glucose and other reagents were commercial grade products.

1.1.6 Statistical processing and test methods

 All measurements are shown as mean soil standard deviation. For comparison between groups, a two-sided test using Student t-test was performed to test for a significant difference. A value of p * 0.05 was considered to be statistically significant, and p * 0.05 was indicated by * and p * 0.01 was indicated by **.

1.2 Experimental results

 1.2.1 Weight change of 0LETF and LET0 rats

 Figure 1 shows the change in body weight of 0LETF rats and LETO rats from 5 weeks to 24 weeks of age. In FIG. 1, —Hataichi indicates the weight change of 0LETF rats, and 110-1 indicates the weight change of LET0 rats.

As shown in Figure 1, no significant difference in body weight was observed between the 0LETF rats and the LET0 rats at the age of 5 weeks, but a significant difference was observed from the age of 6 weeks. Began to appear, and the 0LETF rats were significantly heavier. The weight difference became remarkable as the age of the week progressed.

1.2.2 Changes in glucose tolerance in 0LETF and LET0 rats

 Figure 2 shows the GTT results. In Fig. 2, the band shows the results of 0LETF rats, and the mouth shows the results of LET0 rats.

 As shown in Figure 2, no significant difference in glucose tolerance was observed between the 0LETF rats and the LET0 rats at 5 weeks of age.Blood glucose levels in both groups were below 200 mg / dL, and both groups had glucose tolerance. No dysfunction was observed. At 23 weeks of age, blood glucose levels of four of the six 0LETF rats used in the experiment exceeded 200 mg / dL, and glucose tolerance was significantly worse than that of LETO rats.

1.3 Discussion

 Regarding weight change, a significant difference was detected from 6 weeks of age. In a paper on LETF rats (Ishida, K. et al., Metabolism, 1996. 45 (10) p. 1288-95), it was reported that a significant difference in body weight was observed. It is considered that both 0LETF rats and LET0 rats grew well.

 According to the results of GTT, the glucose tolerance at 5 weeks of age was not so different between 0LETF rats and LETO rats, but at 23 weeks of age, glucose tolerance of 0LETF rats was significantly deteriorated. When lg / kg BW glucose was administered intraperitoneally, if the blood glucose level after one hour exceeded 200 fflg / dL, it was considered to be a symptom of diabetes. Four out of the 0 LETF rats exhibited diabetic symptoms.

From the results of body weight change and GTT, it was confirmed that 0LETF rats showed markedly different growth from LET0 rats and showed diabetes symptoms as they became older. Was done.

2. Expression analysis of diabetes-related genes in OLETF rats

 2.1 Experimental materials and methods

 2.1.1 Animals used

 OLETF rats and LET0 rats whose glucose tolerance was measured in the previous section were used for the next week (6 weeks and 24 weeks). In particular, 24-week-old OLETF rats used were those whose blood glucose level was 200 mg / dL or more 1 hour after administration of 1 g / kg BW glucose.

2.1.2 Experimental procedure

 OLETF rats and LET0 rats were fasted at 6 weeks of age (non-diabetic state) and 24 weeks of age '(oligodiabetic state) from 21:00 for 12 hours, after which whole blood was sampled, after blood removal, liver and muscle Was sampled. RNA is extracted from blood (white blood cells), liver and muscle J¾, and insulin receptor (hereinafter, referred to as “IR”), SH2-containing inositol 5-phosphatase (SH2-containing inos i tol-5-phosphatase> “SHIP2”, Peroxisome Proliferat or -Activated Receptor (PPARa), Calpain 10 (CAM10) ), The relative differences in the expression levels of insulin receptor substrate-U (hereinafter referred to as “IRS-1”) in leukocytes, liver and muscle between 0LETF rats and LET0 rats were analyzed. .

2.1.3 Blood, liver and muscle sampling

OLETF rats and LET0 rats were 6 weeks old (non-diabetic), 24 weeks old (sugar Urinary condition) and fasted for 12 hours from 21:00. After weighing, the rats were anesthetized with ether gas and laparotomy was performed, and sodium heparin (1 OOOU / mL) was injected into the abdominal vena cava using a 27G syringe. At this time, assuming that the whole blood volume of the rat was one-third of the body weight, the required amount of heparin sodium was estimated based on the attached text, and heparin sodium was injected twice as much as the estimated value. After the injection, a falcon tube in which 0.5 mL of a 0.5% EDTA / physiological saline solution had been previously collected was placed under the aorta, and the aorta was cut and blood was collected. The obtained blood sample was immediately subjected to an RNA extraction operation from leukocytes. At the same time, physiological saline was perfused from the portal vein, and the liver and muscle were removed after blood removal. Liver and muscle were immediately immersed in liquid nitrogen after extraction and stored at _80 ° C.

2.1.4 RNA extraction from leukocytes

 RNA extraction from the blood sample obtained in 2.1.3 was performed by a spin column method using QIAamp RNA Blood Mini Kits from QIAGEN. After extracting RNA along the protocol attached to the kit, it was dissolved in DEPC water, the nucleic acid concentration and purity were measured, and stocked at -80 ° C.

 At 6 weeks of age, DNA was found to be in carrier in the aqueous RNA solution.Therefore, RNA extraction at 24 weeks of age was performed using QIAGEN RNase-Free Dnase Digest Set together. Was.

2.1.5 RNA extraction from liver and muscle

The liver and muscle stock obtained in 2.1.3 were broken with a hammer while applying liquid nitrogen. The mixture was transferred to a mortar filled with liquid nitrogen and crushed with liquid nitrogen. From the powdered liver and muscle samples, use Invitrogen's TRIZ0L Reagent to the protocol attached to the reagent. Accordingly, RNA was extracted. RNA was dissolved in DEPC water, nucleic acid concentration and purity were measured, and stocked at -80 ° C.

2.1.6 — Synthesis of single-stranded cDNA

Add 0.2 g of oligo (dT) 12-18 primer (500 M g / mL) to 1 g of total RNA, adjust to 11.5 zL with 0.1% DEPC water, heat denature at 70 ° C for 10 minutes, and leave on ice did. Then, First-Strand Buffer [50 mM tris-HCl (pH 8.3), 75 mM KC1, 3 mM MgCl ₂ ], 10 mM DTT, dNTPs (0.5 mM each), RNase inhibitor (20 U), and SUPERSCRIPT IIRNase Reverse Transcriptase (100 U ) To a total volume of 20 L, and reacted at 42 ° C for 60 minutes. Thereafter, the reaction was stopped by incubating at 94 ° C for 5 minutes to prepare a cDNA sample. The cDNA samples were stored at -20 ° C.

2.1.7 Setting of primer

 From Gene Bank, IR [Goldstein, BJ and AL Dudley, Mol Endocr inol, 1990.4 (2) p.235-44], SHIP2 [Kudo, M. et al., Brain Res Mol Brain Res, 2000.75 (1 ) p.172-7], PPAR r [Guardiola-Diaz, HM et al., J Biol Chem, 1999. 274 (33) p.23368-77], CAPN10 [Ma, H. et al.,〗 Biol Chem, 2001. 276 (30) p.28525-31], IRS-1 [Nature 1991 Jul 4: 352 (6330) p.73-7]> Glyceralde yde 3-phosphate dehydrogenase (GAPDH) [Tso, J, Y. et al., Nucleic Acids Res, 1985. 13 (7) p.2485-502] rat cDNA sequence was extracted and the following primers were designed.

 • Primer set 1 (for IR)

 [PCR product length: 405bp, annealing temperature: 56.4 ° C]

 Upper, 20mer, b position: 3601

5 '-ATCACGTGGTCCGCCTTCTT -3' Lower, 20mer, 3 'pos ii ion: 3986

 5'- TGTCGGAAGAAGCAGTGAAG -3 '

• Primer set 2 (for SHIP2)

[PCR product length: 318bp, annealing temperature: 56.5 ° C] Upper, 20mer, 5 position: 2787

 5'-ATATGGGGAGTGTGTGGTTG -3 '

 Lower, 20mer, 3 'position: 3085

 5'- GGTGGCTTTTCAGGCTCTTC-3 '

• Primer set 3 (for PPARr)

[PCR product length: 484bp, annealing temperature: 55.0 ° C] Upper, 20mer, position: 756

 5 '-GGCCGAGAAGGAGAAGCTGT-3'

 Lower, 20mer, 3 'osition: 1220

 5'_ CATGAATCCTTGTCCCTCTG -3 '

• Primer set 4 (for CAPN10)

[PCR product length: 136 bp, annealing temperature: 60 ° C] Upper, 20mer, 5 position: 282

 5 '-CACCTCCTGGACCAGGTCTT -3'

 Lower, 21mer, 3 'position ion: 417

 5 '-CAAGACAAGGCAGACGGTCAT -3'

• Primer set 5 (for IRS-1)

[PCR product length: 293bp, annealing temperature: 60 ° C] Upper, 20mer, 5 position: 1426

 5'- CGCAGGCACCATCTCAACAA -3 '

 Lower, 20mer, 3 'position: 1699

5'- CATCGTGAAGAAGGCATAGG -3 ' '' Primer set 6 (for GAPDH)

[PCR product length: 296bp, annealing temperature: 58.9 ° C]

 Upper, 20mer, 5 position: 628

 5 '-GCAGCCCAGAACATCATCCC -3'

 Lower, 20mer, 3 'position: 904

 5 '-CCAGCCCCAGCATCAAAGGT -3'

2.1.8 Real-time quantitative PCR

 The amount of mRNA of each gene was quantified by performing Realtime SYBR Green PCR using GAPDH as an internal standard. From cDNA sample 4, supplied enzyme buffer 25 / iL, sense and antisense primers 1 βί each (6Μ each) and sterile purified water 16 / iL: Prepare PCR reaction mixture and make final volume 50 50 L. PCR was performed in the following cycle using ABI PRISM 7700. All samples were measured twice with 50 cycles of heat denaturation at 95 ° C for 15 seconds, annealing at 67 ° C for 5 seconds and amplification at 72 ° C for 10 seconds. Data were calculated using the standard curve method and expressed as relative amounts to GAPDH.

2.1.9 Statistical processing and test methods

 All measurements are shown as mean soil standard deviation. For comparison between groups, a two-tailed test using Student t-test was performed to test for significant differences. Values of p <0.05 were considered statistically significant, and Ρ <0.05 was indicated by ★, and ρ <0, 01 was indicated by ★★.

2.2 Experimental results

2.2.1 Relative mRNA expression in 0LETF and LET0 rats

(i) IR mRNA relative expression

Leukocytes, liver and liver of 6- and 24-week-old 0LETF rats and LET0 rats Table 1 shows the relative amounts of mRNA expression (%) of IR in muscle and muscle. Fig. 3A shows the relative amount (%) of IR mMA expression in leukocytes. In FIG. 3A, the drawing shows the result of 0LETF rat, and the mouth shows the result of LET0 rat. [1]

 6 weeks old LETO OLETF P

White blood cells (n = 6) 100 ± 37 67 ± 15 0.058

Liver (n = 3) 100 ± 8 83 ± 14 0.16

Muscle (n = 3) 100 ± 15 78 ± 14 0.14

24 weeks old L ETO E E T F P

White blood cells (n = 6) 100 ± 14 81 ± 22 0.12

Liver (n = 6) 100 ± 5 79 Sat 17 0.02 *

Muscle (n = 6) 100 ± 38 64 ± 34 0.12 As shown in Table 1 and FIG. 3A, at the age of 6 weeks, in the leukocyte, liver and muscle tissues, between LET0 rats and 0LETF rats No significant difference was detected. At 24 weeks of age, no significant difference was detected between LET0 rats and 0LETF rats in leukocytes and muscles. Was also significantly reduced.

Therefore, even in animals that will develop type 2 diabetes in the future, the mRNA expression levels of leukocytes, liver and muscle IR before the onset of type 2 diabetes are considered to be not significantly different from healthy animals. In animals with type 2 diabetes, the expression of IR mRNA in leukocytes and muscle is not considered to be significantly different from that in healthy animals, but the expression of mRNA in liver IR is more significant than in healthy animals. It is considered that the number has decreased. (ii) SHIP2 mRNA expression relative amount

 Table 2 shows the relative amounts of SHI mRNA expression () in leukocytes and liver of 6- and 24-week-old 0LETF rats and LETO rats. Analysis of SHI mRNA expression in muscle was too small to be analyzed. The relative expression (%) of SHIP2 mRNA expression in leukocytes is shown in FIG. 3B. In FIG. 3B, ■ indicates the result of 0LETF lad, and the mouth indicates the result of LET0 rat.

 [Table 2]

 6 weeks old L ETO PL ETF P

White blood cells (n = 6) 100 ± 19 76 ± 12 0.054

Liver (n = 3) 100 ± 7 84 ± 23 0.33

24 weeks old L ETO PL ETF P

White blood cells (n = 6) 100 ± 32 120 ± 44 0.37

Liver (n = 6) 100 ± 25 87 ± 36 0.49 As shown in Table 2 and FIG. 3B, at the age of 6 weeks and 24 weeks, LET0 rats and 0LETF No significant difference was detected between rats.

 Therefore, even in animals that may develop type 2 diabetes in the future, the expression levels of SHI mRNA in leukocytes and liver before the onset of type 2 diabetes are considered to be not significantly different from healthy animals. In addition, it is considered that there is no significant difference in the expression levels of SHin mRNA in leukocytes and liver in animals with type 2 diabetes compared to healthy animals.

(iii) Relative expression of mRNA of PPART

Leukocytes, liver and liver of 0-LETF and LET0 rats at 6 and 24 weeks of age Table 3 shows the relative expression (%) of PPART mRNA expression in skeletal muscle. FIG. 3C shows the relative amount (%) of PMRT mRNA expression in leukocytes. In FIG. 3C, the image shows the result of the OLETF rat, and the mouth shows the result of the LETO rat.

 [Table 3]

 6 weeks old L E TO OL ETF P

White blood cells (n = 6) 100 ± 39 78 ± 23 0.27

Liver (n = 3) 100 ± 33 120 ± 21 0.4

Muscle (n = 3) 100 ± 39 68 ± 23 0.3

2 4 weeks old L E TO OL ET F P

White blood cells (n = 6) 100 ± 66 119 ± 23 0.34

Liver (n = 6) 100 ± 11 88 ± 19 0.22

Muscle (n = 6) 100 ± 38 50 ± 19 0.0012 ** As shown in Table 3 and FIG. 3C, at the age of 6 weeks, LET0 rats and 0LETF rats in all tissues of leukocytes, liver and muscle No significant difference was detected between them. At 24 weeks of age, no significant difference was detected between LET0 rats and 0LETF rats in leukocytes and liver. It was significantly reduced.

Therefore, even in animals that will develop type 2 diabetes in the future, it is considered that there is no significant difference in the expression levels of PPARr mRNA in leukocytes, liver and muscle before the onset of type 2 diabetes compared with healthy animals. In animals with type 2 diabetes, the expression of PPAR mMA in leukocytes and liver is not considered to be significantly different from that in healthy animals, but the expression of PPARr mRNA in muscle is higher than that in healthy animals. Is also thought to be significantly reduced

(iv) Relative expression of mRNA of CAPN10

 Table 4 shows the relative amounts (%) of CAPN10 mRNA expression in leukocytes, liver and muscle of 6- and 24-week-old OLETF and LETO rats. FIG. 3D shows the relative amount (%) of expression of CAPN10 niRNA in leukocytes. In FIG. 3D, the garden shows the results for OLETF rats, and the mouth shows the results for LETO rats.

 [Table 4]

 L ETO O L E T F P

White blood cells (n = 6) 100 ± 35 48 ± 8 0. .006 **

Liver (n = 3) 100 ± 26 86 ± 18 0, .52

Muscle (n = 3) 100 ± 28 58 Sat 27 0, .14

2 4 weeks old L ETO O L E T F P

White blood cells (n = 6) 100 Sat 36 49 ± 14 0, .0011 **

Liver (n = 6) 100 ± 23 60 ± 11 0 • 004 **

Muscle (n = 6) 100 ± 22 39 ± 18 0, .0004 ** As shown in Table 4 and Figure 3D, at the age of 6 weeks, between the LET0 and 0LETF rats in the liver and muscle Although no significant difference was detected, in leukocytes, the mRNA expression level of 0LETF rats was significantly lower than that of LETO rats. At 24 weeks of age, significant differences were detected between LET0 rats and 0LETF rats in all tissues of leukocytes, liver and muscle.

Therefore, in animals that will develop type 2 diabetes in the future, Although the expression of CAPN10 mRNA in liver and muscle before the onset is not considered to be significantly different from that in healthy animals, the amount of CAPN10 mA expression in leukocytes before onset of type 2 diabetes was significantly reduced compared to healthy animals. It is thought that there is. Further, it is considered that the amount of CAPN10 mRNA expression in leukocytes, liver and muscle is significantly reduced in animals developing type 2 diabetes compared to healthy animals.

(v) Relative mRNA expression of IRS-1

 Table 5 shows the relative amount (%) of IRS-1 mRNA expression in leukocytes, liver and muscle of 6- and 24-week-old 0LETF rats and LETO rats. FIG. 3E shows the relative amount (%) of CAPN10 mRNA expression in leukocytes. In addition, in FIG. 3E, the drawing shows the result of 0LETF rat and the mouth shows the result of LET0 rat.

 [Table 5]

 6 weeks old L ETO O L ET F P

White blood cells (n = 6) 100 ± 31 66 ± 19 0.03 *

Liver (n = 3) 100 ± 11 72 ± 21 0.12

Muscle (n = 3) 100 ± 54 72 ± 27 0.48

2 4 weeks old L ETO OL E T F P

White blood cells (n = 6) 100 Sat 31 46 ± 18

Liver (n = 6) 100 ± 16 83 Sat Π 0.13

Muscle (n = 6) 100 ± 79 50 ± 46 0.22 As shown in Table 5 and Figure 3E, at the age of 6 weeks, a significant difference was detected between LET0 rats and 0LETF rats in liver and muscle. However, in leukocytes, the mRNA expression level of 0LETF rats was lower than that of LET0 rats. Was significantly reduced. At 24 weeks of age, no significant difference was detected between LET0 rats and 0LETF rats in liver and muscle. Was also significantly reduced.

 Therefore, in animals that will develop type 2 diabetes in the future, the amount of IRS-1 mRNA expression in liver and muscle before the onset of type 2 diabetes is not considered to be significantly different from that in healthy animals, but before the onset of type 2 diabetes. It is considered that the expression level of IRS-1 mRNA in leukocytes is significantly lower than that in healthy animals. In animals with type 2 diabetes, the amount of IRS-1 mRNA expression in liver and muscle is considered to be not significantly different from that in healthy animals, but the amount of IRS-1 mRNA expression in leukocytes is It is thought that it is significantly reduced compared to healthy animals.

2.2.2 Summary of gene expression changes

 In animals that will develop type 2 diabetes in the future (0LETF rats), CAPN10 and IRS-1 mRNA expression levels in leukocytes before (6 weeks) and after (24 weeks) the onset of diabetes Was lower than that in healthy animals (LET0 rats), indicating that the expression levels of CAPN10 and IRS-1 genes in leukocytes were lower than the normal expression levels as diabetes progressed .

Therefore, it is considered that diabetes can be diagnosed by using the expression levels of the CAPN10 gene and the IRS-1 gene in leukocytes as indices. That is, when the expression levels of the CAPN10 gene and the IRS-1 gene in leukocytes are lower than the normal expression levels, it is possible that diabetes will develop in the future or that diabetes is currently occurring. It is considered possible to diagnose. Industrial potential

 According to the present invention, first, type 2 diabetes can be diagnosed by analyzing gene expression in tissues that can be easily sampled (especially, before the onset of type 2 diabetes, whether or not it is possible to develop type 2 diabetes in the future) Can be diagnosed to enable early detection of type 2 diabetes), and a method and kit for diagnosing type 2 diabetes are provided. Further, according to the present invention, secondly, a substance having a preventive / therapeutic effect on type 2 diabetes can be screened by analyzing gene expression in a tissue that is easily sampled, thereby preventing / treating the type 2 diabetes. And a screening kit for a substance having the following.

Claims

The scope of the claims 1. A method for diagnosing type 2 diabetes, comprising diagnosing type 2 diabetes using the expression level of the CAPN10 gene or IRS-1 gene in leukocytes collected from the blood of the subject as an index.  2. The diagnostic method according to claim 1, wherein the expression level is measured based on the abundance of mRNA encoding CAPN10 or IRS-1.  3. The diagnostic method according to claim 1, wherein the expression level is measured based on the abundance of CAPN10 or IRS_1.  4. When the expression level of CAPN10 gene or IRS-1 gene in leukocytes of the subject is lower than the expression level of CAPN10 gene or IRS-1 gene in leukocytes of a healthy subject, 2. The diagnostic method according to claim 1, wherein the diagnosis is made that the individual has a possibility of developing type 2 diabetes in the future or that the subject has a possibility of developing type 2 diabetes at present.  5. A kit for diagnosing type 2 diabetes, comprising an oligonucleotide or polynucleotide capable of hybridizing to a nucleic acid encoding CAPN10 or IRS-1.  6. A kit for diagnosing type 2 diabetes, comprising an antibody capable of reacting with CAPN10 or IRS-1 or a fragment thereof. 7. After administering the candidate substance to a type 2 diabetes model animal, using the expression level improving effect of the CAP N10 gene or the IRS_1 gene in leukocytes collected from the blood of the animal as an index, the type 2 diabetes of the candidate substance A method for screening a substance having a preventive and therapeutic effect on type 2 diabetes, which comprises determining the preventive and therapeutic effects. 8. A kit for screening for a substance having a preventive and / or therapeutic effect on type 2 diabetes, comprising an oligonucleotide or a polynucleotide capable of hybridizing to a nucleic acid encoding CAPN10 or IRS_1.  9. A screening kit for a substance having a preventive / therapeutic effect for type 2 diabetes, comprising an antibody or a fragment thereof that can react with CAPN10 or IRS-1.