WO2007066794A1

WO2007066794A1 - Brak-knockout nonhuman animal

Info

Publication number: WO2007066794A1
Application number: PCT/JP2006/324622
Authority: WO
Inventors: Takahiko Hara; Noriko Nara; Yuki Terasawa; Chouji Taya; Hiroshi Shitara; Rie Ishii
Original assignee: Tokyo Metropolitan Organization For Medical Research
Priority date: 2005-12-06
Filing date: 2006-12-05
Publication date: 2007-06-14

Abstract

It is intended to provide a BRAK-knockout nonhuman animal. This knockout nonhuman animal is characterized in that the BRAK gene has been disrupted therein.

Description

Specification

BRAK knockout non-human animal technical field

The present invention relates to a knockout non-human animal in which the BRAK gene has been disrupted, a method for screening for a BRAK antagonist using the same, and a method for testing the side effects of a BRAK antagonist. Background technology

In recent years, the number of patients with diabetes, which is closely related to obesity (especially type 2 diabetes, which shows insulin resistance), has been increasing worldwide. Along with this, hypertension and arteriosclerosis are also on the rise. The onset mechanism of these conditions is thought to be due to a comprehensive failure of fat metabolism and hormonal regulation, and they have come to be collectively known as metabolic syndrome.

Recently, gene expression analysis using white adipose tissue from obese mice and obese patients has shown that macrophages in white adipose tissue play an important role in the induction of obesity and associated insulin resistance. reported (Weisberg SP et al., J. Clin. Invest., vol. 112, p. 1796- 1808, 2003; Xu H et al., J. Clin. Invest., vol. 112, p. 1821-1830 , 2003). In experiments using obese mice, these macrophages produced TNF, which inhibited insulin-induced glucose absorption. At the same time, secretion of inflammatory cytokines such as IL-1 and IL-6 increased, leading to a chronic inflammatory state (Wellen KE et al., J. Clin. Invest" vol. 112, p. 1785- 1788, 2003; Wellen KE et al., J. Clin. Invest., vol. 115, p. 1111- 1119, 2005).In fact, in mice in which TNF or its receptor was disrupted, obesity-induced Insulin resistance was improved (see Uysal KT et al., Nature, vol. 389, p. 610-614, 1997).

Recent studies have suggested that activation of JNK kinase and the endoplasmic reticulum stress signaling pathway is essential for obesity-induced insulin resistance. (See Hirosumi J et al., Nature, vol. 420, p. 333-336, 2002; Ozcan U et al., Science, vol. 306, p. 457-461, 2004). It was also reported that blocking the ΙΚΚ·3/NF-κB pathway in the liver ameliorated obesity-induced insulin resistance (Cai D et al" Nat. Med., vol. 11, p. 183- 190, 2005; see Arkan MC et al" Nat. Med., vol. 11, p. 191-198, 2005). JNK is also activated by IKK. Therefore, the endoplasmic reticulum stress/ΙΚΚ/JK pathway is activated by increased secretion of inflammatory cytokines from obese adipose tissue caused by macrophage infiltration and fatty acid loading. This activation is thought to be the main cause of insulin resistance associated with obesity.

Furthermore, it has been reported that the production of adiponectin, an important secretory hormone that controls the energy metabolism of lipids and carbohydrates, is suppressed in the adipose tissue of obese mice (Yamauchi T et al., Nat. Med). ., vol. 7, p. 941-946, 2001).

By the way, ``BRAK:'' (Breast and Kidney expressed chemokine) was cloned by Hromas et al. in 1999 as a new human chemokine gene whose expression is lost in tumor cell lines. Biochem. Biophys. Res. Commun., vol. 255, p. 703-706, 1999). Subsequently, multiple groups have reported analytical results regarding the same chemokine molecules (Frederick MJ et al., Am. J. Pathol., vol. 156, p. 1937-1950, 2000; Cao X et al. al., J. Immunol., vol. 165, p. 2588-2595, 2000; Sleeman MA et al., Int. Immunol., vol. 12, p. 677-689, 2000).

To date, over 50 chemokine genes have been found to be encoded in the chromosomes of mammals. These chemokines are composed of 92-99 amino acids (molecular weight approximately 8,000-10,000), each having four cysteine residues that are disulfide-bonded. This cysteine residue and the surrounding amino acid sequence are characteristic of the N-terminal side of chemokines, and based on this characteristic, chemokines are classified into "CC", "CXC", "CX ₃ C", and " (See Zlotnik A et al., Immunity, vol. 12, p. 121-127, 2000). Among them, 16 types of CXC chemokines have been identified so far, and the ELR + Classified into CXC chemokines and ELR—CXC chemokines. BRAK belongs to the ELR-CXC chemokines that do not have an ELR sequence, and has been named ``CXCL14'' to unify the name.

Recent studies have shown that BRAK has the ability to specifically migrate human peripheral blood monocyte-derived macrophages activated by prostaglandin _E2 (Kurth I et al., J. Exp. Med., vol. 194, p. 855-861, 2001). BRAK is also expressed in dendritic progenitor cells differentiated from human monocytes and hematopoietic progenitor cells (Shellenberger TD et al., Cancer Res., vol. 64, p. 8262-8270, 2004; Schaerli P et al., Immunity , vol. 23, p. 331-342, 2005) and human breast cancer-derived epithelial cell lines (see Allinen M et al., Cancer Cell, vol. 6, p. 17-32, 2004). , was reported to have specific migratory activity. Thus, BRAK is a unique chemokine that has migratory activity not against T cells and B cells, but against tissue phages and dendritic progenitor cells (Kurth I et al., J Exp. Med., vol. 194, p. 855-861, 2001), and no other CXC chemokines are known to have the same spectrum of action.

In humans, BRAK expression has been confirmed in the small intestine, kidney, liver, uterus, and mammary gland (see Sleeman MA et al., Int. Immunol., vol. 12, p. 677-689, 2000). An interesting finding has been obtained that BRAK expression is lost in uterine cancer and breast cancer patient samples or cancer cell lines (Hromas R et al., Bibchem. Biophys. Res. Commun., vol. 255, p. 703-706, 1999; see Frederick MJ et al., Am. J. Pathol., vol. 156, p. 1937 1950, 2000). Based on this finding, it is speculated that BRAK-responsive cells are less likely to migrate to cancer tissues (see Shurin GV et al., J. Immunol., vol. 174, p. 5490-5498, 2005). On the other hand, in adult mice, the expression of BRAK has been mainly confirmed in the lungs, skeletal muscles, fat and ovaries (Sleeman MA et al., Int. Immunol., vol. 12, p. 677-689, 2000 reference) . However, no knowledge has yet been obtained regarding the behavioral entities and functions of BRAK-responsive cells, particularly monocytes and macrophages in white adipose tissue, in individual mice. Disclosure of invention

Therefore, the problem that the present invention aims to solve is to clarify the relationship between monocyte and macrophage chemotaxis to white adipose tissue and BARK, and to investigate the dynamics of various parameters related to obesity and insulin response. The objective is to provide knockout non-human animals in which the BRAK gene has been disrupted for analysis. Furthermore, another object of the present invention is to provide a method for screening BRAK antagonists and a method for testing the side effects of BRAK antagonists using this non-human animal.

The inventors of the present invention conducted extensive experiments to solve the above problems. As a result, we created BRAK knockout mice (BRAK homozygous-deficient mice (-/_)), and these knockout mice survived for a long period of time after birth without exhibiting abnormal characteristics, and the BRAK gene was deleted at the protein expression level. I confirmed that it was. We then used these knockout mice and littermate control mice (wild-type mice (+/+), BRAK heterozygous mice (+/-)) in an obese state to investigate insulin resistance and macrophages in white adipose tissue. By comparing and evaluating the number of oral phages, etc., we discovered that there was a clear difference between the two mice, leading to the completion of the present invention. - That is, the present invention is as follows. .

(1) Knockout non-human animal with disrupted BRAK gene.

Examples of the above-mentioned non-human animals include those that do not show insulin resistance when made obese, and those that have a reduced number of macrophages in white adipose tissue when made obese. .

Furthermore, examples of the above-mentioned non-human animals include rodents, and examples of the rodents include mice.

(2) A method for screening BRAK antagonists, in which the non-human animal described in (1) above is used as a control non-human animal, and non-human animals other than the control non-human animal are used as test subjects. a step of making the control non-human animal and the test non-human animal obese, a step of administering a candidate substance to the test non-human animal, and a step of making the control non-human animal and the test non-human animal obese. The method described above, comprising the step of comparatively evaluating the obese state of the animal. (3) A method for testing the side effects of BRAK antagonists, in which the non-human animal described in (1) above is used as a control non-human animal, and a non-human animal other than the control non-human animal is used as a test non-human animal. 2. The method described in s above, comprising the steps of: administering a BRAK antagonist to the non-human test animal; and comparing and evaluating the control non-human animal and the non-human test animal. Brief description of the drawing

Figure 1A is a diagram regarding the establishment of a BRAK knockout mouse line. The structure of the targeting vector and the restriction enzyme recognition site of the mouse BRAK gene locus after homologous recombination are schematically shown. The black box (white text) indicates the exon part of the BRAK gene. P1 and P2 indicate the positions of PCR primers used to identify recombinant ES cells.

Figure 1B is a diagram regarding the establishment of a BRAK knockout mouse line. The spleen genomic DNA of wild-type mice (+/+), heterozygous mice (+/_), and homozygous mice (-/-) was digested with the restriction enzyme EcoRI, and then analyzed by Southern blotting.

Homologous recombination at the BRAK gene locus was confirmed.

Figure 1C is a diagram regarding the establishment of a BRAK knockout mouse line. Total RNA was extracted from the brains, lungs, and muscles (skeletal muscles) of wild-type mice (+/+), heterozygous mice (+/_), and homozygous mice (_/-), and Northern The expression level of BRAK mRNA was assayed by blotting. /3 -actin is a control that indicates the amount of RNA loaded on each lane.

FIG. 2A is a diagram regarding the characteristics of each mouse genotype. The abdomens of wild type (+/+), heterozygous (+/-), and homozygous (-/_) male mice born from the same litter are shown.

Figure 2B is a diagram regarding the characteristics of each mouse genotype. Female mice of each genotype were fed a high-fat diet and their body weights were measured daily over 14 weeks. FIG. 2C is a diagram regarding the characteristics of each mouse genotype. The ``body weight'' of female mice of each genotype raised on normal diet (n=6 each) or high-fat diet (n=5 each) is shown. The graph shows the mean and standard error of each group. Figure 2D is a diagram regarding the characteristics of each mouse genotype. The daily intake per body weight of female mice of each genotype is shown when they are fed a normal diet (n=6 each) or a high-fat diet (n=5 each). The graph shows the mean value ± standard error of each group.

FIG. 2E is a diagram regarding the characteristics of each mouse genotype. The organ weight ratios are shown when female mice of each genotype are fed a normal diet (n=6 each) or a high-fat diet (n=5 each). The graph shows the mean and standard error of each group.

Figure 2F is a diagram regarding the characteristics of each mouse genotype. Mac 1 negative isolated from white fat around the uterus (Mac) when female mice of each genotype were fed normal diet (n = 6 each) or high fat diet (n = 5 each) and the number of Mac 1 positive (Mac 1 ⁺ ) cells. The graph shows the mean and standard error of each group.

Figure 3 is an image of mouse white adipose tissue. Paraffin sections of periuterine white adipose tissue were prepared from heterozygous (A, C) and homozygous (B, D) female mice fed normal diet (A, B) or high-fat diet (D). Then, immunostaining was performed using anti-F4/80 antibody as the primary antibody. Brown signals indicate the locations of F4/80-positive cells. FIG. 4A is a diagram regarding blood glucose levels of mice of each genotype. Normal feed [ (+/+) (+/_ ), n=7 ; (-/-) , n=6], or high-fat feed [ (+/+) (+/-) , n=8 ; ( -/-), n=6], and their blood sugar levels were measured after fasting overnight. The graph shows the mean and standard error of each group.

Figure 4B is a diagram related to the insulin resistance test of mice of each genotype.Female mice of each genotype fed _a normal diet (RD) or a high-fat diet (HFD, 16 weeks) were fasted overnight. After that, insulin was injected intraperitoneally. Blood sugar levels (mg/dl) after insulin administration were measured over time. Changes in blood sugar levels were compared to sisters born in the same litter [normal feed: (+/+) (+/-), n=7; (-/-), n=6, high-fat feed: (+/+) (+/ -), n=8; (-/-), n=5] and expressed as mean value and standard error.

Figure 4C is a diagram regarding the glucose tolerance test (glucose uptake rate) of each mouse genotype. Female mice fed a normal diet (RD) or a high-fat diet (HFD, for 16 weeks) were fasted overnight and then intraperitoneally injected with glucose. Blood sugar levels (mg/dl) were measured over time after glucose administration. Changes in blood sugar levels were compared to sisters born in the same litter [normal feed: (+/+) (+/-), n=7; (-/-), n=6, high-fat feed: (+/+) (+/ -) , n=8 ; (-/-), n=6] and expressed as the mean value and standard error.

FIG. 5A is a diagram regarding hormone secretion levels of mice of each genotype. Serum from female mice of each genotype raised on normal diet (n=6 each) or high-fat diet (n=5 each) was collected, and the insulin content was quantified using ELISA. The graph shows the mean and standard error of each group.

FIG. 5B is a diagram regarding hormone secretion levels of mice of each genotype. Serum from female mice of each genotype raised on normal diet (n = 6 each) or high fat diet (n = 5 each) was collected, and the leptin content was quantified using ELISA. The graph shows the mean and standard error of each group.

FIG. 5C is a diagram regarding hormone secretion levels of mice of each genotype. Serum from female mice of each genotype raised on normal diet (n=6 each) or high-fat diet (n=5 each) was collected, and the content of adiponectin was quantified using ELISA. Daraf indicates the mean and standard error of each group.

Figure 5D is a diagram regarding hormone secretion levels of each mouse genotype. The serum cholesterol concentration of mice raised on a high-fat diet [(+/+) (+/-), n=6; (-/-), n=5] was determined using an enzymatic assay kit. The graph shows the mean and standard error of each group.

FIG. 5E is a diagram regarding hormone secretion levels of each mouse genotype. The serum triglyceride concentration of mice raised on a high-fat diet [(+/+) (+/-), n=6; (-/_), n=5] was determined using an enzymatic assay kit. The graph shows the mean and standard error of each group.

FIG. 5F is a diagram regarding hormone secretion levels of each mouse genotype. Serum free fatty acid concentrations of mice raised on a high-fat diet [(+/+) (+/-), n=6; (_/-), n=5] were determined using an enzymatic assay kit. The graph shows the mean and standard error of each group. FIG. 6A is a diagram regarding the expression of BRAK mRNA. The expression of BRAK mRNA in each organ of adult mice was analyzed by Northern plotting. The /3-actin probe is a control that shows the amount of RNA loaded on each lane.

FIG. 6B is a diagram regarding induction of BRAK mRNA expression in obese mice. Uterine periphery of female mice of each genotype fed normal diet (ND) or high-fat diet (HFD) The expression of BRAK mRNA in white adipose tissue was analyzed by Northern blotting. /3 -tubulin probe is a control that shows the amount of RNA loaded on each lane. Figure 7A shows the establishment of MCK-BRAK transgenic mice. Total RNA was extracted from the skeletal muscles of C57BL/6 mice and MCK-BRAK transgenic mice, and BRAK mRNA expression was detected by Northern blotting. It was tested by testing. ]3 -actin is a control showing the amount of RNA loaded on each lane.

FIG. 7B is a diagram regarding insulin resistance test of each mouse genotype. Female mice of each genotype fed a high-fat diet (HFD for 12 weeks) were fasted overnight and then intraperitoneally injected with insulin. Blood sugar levels (mg/dl) were measured over time after insulin administration. Changes in blood sugar levels were measured for 3 animals in each group, and expressed as mean value and standard error.

Tg indicates transgenic mouse.

FIG. 7C is a diagram regarding the insulin resistance test of each mouse genotype. Female mice of each genotype kept on normal diet (RD) were fasted overnight and then intraperitoneally injected with insulin. ' Blood sugar levels (mg/dl) after insulin administration were measured over time. Changes in blood sugar levels were measured in units of [(+/_), (_/-)Tg, n=6; (-/-), n=3] and expressed as mean value and standard error.

Figure 7D shows that BRAK partially inhibits insulin signaling. Differentiation-induced C2C 12 cells were stimulated with the indicated cytokines, and phosphorylation of AJct Se ⁷³ was measured by Western blotting. BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be explained in detail below. The scope of the present invention is not limited to these explanations, and modifications other than the following examples may be made as appropriate without departing from the spirit of the present invention.

Note that this specification includes the entirety of the specification of Japanese Patent Application No. 2005-352519, which is the basis of the priority claim of this application. In addition, all publications cited in this specification, such as prior art documents, publications, patent publications, and other patent documents, are incorporated herein by reference. 1. Overview of the invention

It has been suggested that macrophages are involved in the increase in white adipose tissue associated with obesity.The detailed mechanism has not yet been elucidated. The present inventors created knockout mice in which the BRAK (Breast and Kidney expressed chemokine)/CXCL14 gene (SEQ ID NO: 1), a tissue macrophage chemotactic factor, was disrupted. These BRAK homozygous-deficient mice [(-/-)] had lower average body weight and lower amount of white fat around the genitals than their control littermates [(+/+), (+/-)]. .

Next, when both mice were raised on a high-fat diet, the BRAK homozygous-deficient mice had a slower rate of weight gain and a significantly higher number of macrophages in their white adipose tissue than their littermate control mice. It was small. In addition, BRAK homozygous-deficient mice did not exhibit insulin resistance or a decrease in adiponectin secretion, which normally occurs in obese mice fed a high-fat diet. Furthermore, there was no significant increase in blood insulin levels that occurs in type 2 diabetic mice. Furthermore, it was confirmed that BRAK mRNA is also expressed in white adipose tissue, and that its expression level increases markedly in obese mice.

The above experimental results showed that BRAK is a novel mediator substance that induces obesity-induced insulin resistance. BRAK homozygous-deficient mice do not exhibit obesity-induced insulin resistance due to defective migration of activated macrophages to white adipose tissue, and exhibit resistance to type 2 diabetes, making them extremely useful as a new type of genetically modified animal (model animal). It is something.

2. Non-human animals

In the present invention, a knockout non-human animal in which the BRAK gene has been disrupted refers to a non-human animal that has been disrupted due to disruption, deletion, substitution, etc., of all or part of the endogenous gene of the non-human animal that encodes BRAK shown in SEQ ID NO: 1. BRAK refers to a non-human animal that has been activated and has lost the ability to express BRAK. In other words, the non-human animal of the present invention can be said to be a non-human animal in which the function of the BRAK gene has been lost on the chromosome. Specifically, the non-human animal of the present invention refers to a non-human animal having a BRAK homozygous deletion genotype [(-/-)], and a wild-type genotype [(+/+)] and BRAK heterozygous deficiency genotype Non-human animals of the same species with [(+/_)] are excluded.

The non-human animal of the present invention is preferably one born according to Mendel's law. In this case, the non-human animal of the present invention and a control non-human animal from the same litter (i.e. (+Λ) and (+/ This method has the advantage that it is possible to obtain non-human animals (-), and to conduct accurate comparative experiments using these.

Here, non-human animals that can be used in the present invention include mammals other than humans, such as mice, rats, guinea pigs, pigs, dogs, cats, monkeys, cows, cows, and horses. Among these, rodents (Rodentia) such as mice, rats, and guinea pigs are preferred, and mice are more preferred.

Specifically, the non-human animal of the present invention does not exhibit insulin resistance when made obese. Furthermore, the non-human animal of the present invention is one in which the number of macrophages in white adipose tissue is reduced when the animal is rendered obese. As mentioned above, based on the results of genetic analysis using white adipose tissue from obese mice and obese patients, we believe that macrophages attracted to white adipose tissue are significantly involved in the insulin resistance associated with obesity. It is being Therefore, in the non-human animal of the present invention, the number of macrophages in white adipose tissue is significantly reduced when the animal is made obese by losing the ability to express BAE, which has migratory activity against macrophages, and as a result, It has the characteristic that it does not show insulin resistance even when obese, and does not develop type 2 diabetes. Here, obese state means that when a non-human animal is fed a high-fat feed, its body weight is 1.2 times or more (preferably) compared to a non-human animal of the same genotype fed a normal feed. 1.5 times or more, more preferably 1.8 times or more, even more preferably 2 times or more). Obese mice can be obtained, for example, by feeding female mice aged 6 weeks or older a high-fat diet such as HFD32 (manufactured by Nippon Clea Co., Ltd.) for 12 weeks or more, but there are limitations. Not done.

Below, the method for producing a non-human animal of the present invention will be explained using a case where the non-human animal is a mouse as an example. Even when the non-human animal is other than a mouse, the non-human animal can be created based on the explanation below and the common general knowledge in the field.

The method for creating BRAK knockout mice (BRAK homozygous deletion mice) is as follows: Any known method may be used as long as it is capable of producing knockout mice that have lost the ability to express BRAK, and the method is not limited. As a specific method of production, the following procedure can be exemplified.

That is, first, a gene fragment is obtained from a mouse gene library by a method such as PCR, and this gene fragment is used to screen the BRAK gene (or a portion thereof). Next, using recombinant DNA technology, part or all of the screened BRAK gene is replaced with a marker gene, such as a neomycin resistance gene, and furthermore, a marker gene such as diphtheria toxin (diphtheria toxin) is added to the 5' end. DT) gene, diphtheria toxin A fragment (DT-A) gene, or herpes simplex virus thymidine kinase (HSV-tk) gene, etc., are introduced to create a tagging vector. . After the created targeting vector is linearized, it is introduced into ES cells by electroporation, etc., and homologous recombination is performed. From the obtained homologous recombinants, ES cells exhibiting resistance to biological substances such as G418 and ganciclovir (GA C) are selected. Here, it is preferable to confirm whether the selected ES cells are the desired recombinant cells by Southern blotting or the like.

Next, the above recombinant ES cells are microinjected into a mouse morula or blastocyst, and the morula or blastocyst is returned to the foster parent mouse to create a germline chimeric mouse. do. By crossing this chimeric mouse with a wild-type Mavus, a heterozygous mouse (BRAK heterozygous mouse) can be obtained, and further, by crossing these heterozygous mice with each other, a BRAK knockout mouse (BRAK homozygous mouse) can be obtained. deficient mice) can be obtained. In this way, littermate control mice (wild-type and heterozygous mice) are obtained along with the BRAK knockout mice, according to Mendel's rules.

In addition, regarding the production method according to the above example, "Mouse Lab Manual 2nd Edition - Experimental Methods in the Post-Genome Era - (pp. 271-296)" (Publication date: May 26, 2003 (first edition), Edited by: This can be carried out by appropriately referring to the method described in the Tokyo Metropolitan Institute of Clinical Medicine, Experimental Animal Research Division, Publisher: Terumasa Hirano, Publisher: Springer Verlag Tokyo Co., Ltd.). Methods for confirming that the BRAK gene function has been lost on the chromosome in the resulting BRAK knockout mice include, for example, isolating DNA from the spleen of the mouse and confirming by Southern blotting, etc. Examples include a method in which RNA is isolated from the brain, lungs, etc. of a boar mouse and confirmed by Northern blotting, etc., or a method in which BRAK expression in the mouse is confirmed by Western blotting, etc. '

In addition, to confirm that the obtained BRAK knockout mice do not show insulin resistance when made obese, for example, first make them obese by ingesting a high-fat diet, and then After the mice have been fasted for a specified period, insulin is injected intraperitoneally. Thereafter, when the blood sugar level is monitored over time, there is a method to confirm that the blood sugar level remains significantly lower than that of the control mouse (preferably a control mouse from the same litter). Can be mentioned. '

Furthermore, to confirm that the obtained BRAK knockout mice have a reduced number of macrophages in white adipose tissue when made obese, for example, first make them obese by ingesting a high-fat diet. A mouse is used, and the white adipose tissue of this mouse is collected and finely fragmented. Next, after dissolving the fragmented tissue with various enzymes and solutions, only macrophages are fluorescently labeled, and the number of macrophages is measured by flow cytometry or a cell sorter. Note that fluorescent labeling of macrophages is preferably carried out using, for example, a piotinylated anti-macrophage marker antibody or streptavidin labeled with a fluorescent substance such as FITC.

3. Screening method etc.

(1) Screening method for BRAK fantagonists

As mentioned above, the non-human animal of the present invention lacks the ability to migrate toward tissue macrophages due to the loss of the ability to express BRAK. As a result, it has characteristics such as insulin non-resistance and a reduction in the number of macrophages in white adipose tissue. Therefore, a non-human animal other than the non-human animal of the present invention (a non-human animal that has not lost the ability to express BRAK (non-human test animal)) is used, and an antagonist candidate substance is added to it. and a BRAK antagonist. try cleaning. In other words, if characteristics similar to those of the non-human animal of the present invention (control non-human animal) are observed in the test non-human animal after administration of the candidate substance,

This means that even though BRAK is expressed, BRAK activity is suppressed. Therefore, the administered candidate substance can be determined to be a BRAK antagonist. That is, the present invention provides a method for preparing a BRAK antagonist using the non-human animal of the present invention (control non-human animal) and a non-human animal other than the non-human animal of the present invention (test non-human animal). Includes screening methods.

Here, the term BRAK antagonist refers to a substance that binds to the BRAK receptor in monocytes and macrophages and inhibits the migratory activity of BRAK, but does not itself exert any effect due to the binding.

Specifically, the screening method is characterized by including the following steps (i) to (iii). Note that steps (1) and (ii) may be performed in any order, and may be performed first or at the same time. (i) A process of making the control non-human animal and the test non-human animal obese (obesity process) (ii) A process of administering the candidate substance to the test non-human animal (administration process)

(ιϋ) Step of comparatively evaluating the obesity status of the control non-human animal and the test non-human animal (Evaluation step) Each of these steps will be explained below.

(1) Obesity process

The test non-human animal is an animal of the same species as the non-human animal of the present invention that serves as the control non-human animal, and the BRAK gene has not been disrupted (the BRAK gene function has been lost on the chromosome). In particular, wild-type and/or BRAK hetero-deficient animals can be used. The test non-human animal is preferably an animal from the same litter as the non-human animal of the present invention (BRAK homozygous non-human animal).

Obesity in this process means that by feeding a non-human animal a high-fat feed, its body weight is 1.2 times greater than that of a non-human animal of the same genotype fed a normal feed. or more (preferably 1.5 times or more, more preferably 1.8 times or more, even more preferably 2 times or more). Obese mice can be obtained, for example, by feeding female mice 6 weeks of age or older a high-fat diet such as HFD32 (manufactured by Nippon Clea Co., Ltd.) for 12 weeks or more, but there are limitations. Not done.

When making a control non-human animal and a test non-human animal obese, it is preferable that the feed intake conditions be the same for both animals.

(ϋ) Administration process

Candidate substances (BRAK antagonist candidates) to be administered to non-human test animals include various naturally occurring or artificially synthesized peptides, proteins (including antibodies), polynucleotides, and low-molecular organic compounds. I can give an example.

The candidate substance can be administered orally or parenterally, and there are no limitations, and in either case, known administration methods and conditions can be employed. The dosage can also be determined as appropriate, taking into account the type and condition of the non-human animal being tested, the type of candidate substance, etc.

(iii) Evaluation process

In this step, the comparative evaluation target includes, for example, at least one selected from the group consisting of insulin resistance, the number of macrophages in white adipose tissue, and adiponectin concentration in serum. BRAK antagonists can be easily screened by comparing these evaluation targets between control non-human animals and test non-human animals made obese.

Examples of each comparative evaluation method will be explained below.

In this evaluation method, first, control non-human animals and test non-human animals (hereinafter referred to as each non-human animal) are fasted for a predetermined period of time (for example, 16 hours in the case of mice), and blood sugar levels are determined. After stabilization, the insulin solution is injected intraperitoneally. Thereafter, blood sugar levels are compared between the control non-human animal and the test non-human animal when blood sugar levels are monitored over time. At this time, if there is no significant difference between the two animals, the administered candidate substance can be evaluated as the desired BRAK antagonist. On the other hand, if the test non-human animal maintains a significantly higher level of It can be evaluated that it is not a BRAK antagonist.

Comparative evaluation of the number of phages in white adipose tissue>

In this evaluation method, first, white adipose tissue from each non-human animal is collected and finely fragmented. Next, after dissolving the fragmented tissue with various enzymes and solutions, only the macrophages are fluorescently labeled. The number of macrophages is measured using flow cytometry or a cell sorter, and the measured number is compared between the control non-human animal and the test non-human animal. At this time, if there is no significant difference between the two animals, the administered candidate substance can be evaluated to be the desired BRAK antagonist. On the other hand, if the number of measurements was significantly higher in the non-human test animals, it can be evaluated that the administered candidate substance is not the intended BRAK antagonist. Note that the explanation in 2. above can be applied to fluorescent labeling of macrophages.

In this evaluation method, various known measurement kits (eg, mouse adiponectin ELISA kit (manufactured by Otsuka Pharmaceutical Co., Ltd.)) can be used. The measured serum adiponectin concentration is compared between the control non-human animal and the test non-human animal. At this time, if there is no significant difference between the two animals, the administered candidate substance can be evaluated as the desired BRAK antagonist. On the other hand, if the concentration was significantly lower in the non-human test animals, it can be assessed that the administered candidate substance is not the intended BRAK antagonist.

For further details of the various comparative evaluation methods exemplified above, the methods and conditions described in Examples described later can be referred to as appropriate.

The screening method may include other steps in addition to the various steps described above, and is not limited. (2) Method for testing side effects of BRAK antagonists

Even if the BRAK antagonist obtained by the screening method described in (1) above shows excellent properties as an antagonist, it may be considered useful if it has adverse side effects on the animals to which it is administered. Therefore, testing for such side effects is important. The non-human animal of the present invention has the same genetic traits as the wild type, except that it is deficient in the BRAK gene, and can survive for a long period of time after birth without exhibiting any abnormal characteristics. Therefore, when a BRAK antagonist is administered to a non-human animal other than the non-human animal of the present invention (test non-human animal), and BRAK is rendered non-functional in the same way as the non-human animal of the present invention, The condition after administration is compared with that of the non-human animal of the present invention (control non-human animal). At this time, if a symptom (adverse effect) is observed in the test non-human animal that is not present in the control non-human animal, it can be determined that the BRAK antagonist has a side effect related to the symptom. Examples of side effects include effects on survival rate and weight gain, and effects on hematopoietic function and reproductive function, and these can be used as indicators for testing side effects.

In other words, the present invention provides a method for determining the side effects of a BRAK antagonist using the non-human animal of the present invention (control non-human animal) and a non-human animal other than the non-human animal of the present invention (test non-human animal). Also includes testing methods. Here, the BRAK antagonist is as described in (1) above, and is preferably obtained by the screening method described in (1) above.

Specifically, the testing method is characterized by including the following steps ω and (ϋ).

(i) The process of administering the BRAK antagonist to the test non-human animal (administration process) (ϋ) The process of comparing and evaluating the control non-human animal and the test non-human animal (evaluation process) Each of these steps is described below. Explain.

(Administration process

The test non-human animal to which the BRAK antagonist is administered is preferably obese, but is not limited thereto. Regarding the non-human test animal and the obese state, the same explanation as "(i) Obesity step" in the screening method (1) above can be applied.

Administration of the BRAK antagonist can be carried out orally or parenterally, without limitation, and in either case, known administration methods, administration conditions, etc. can be employed. The dosage also depends on the type and condition of the non-human animal being tested, as well as the species of the BRAK antagonist. It can be set as appropriate, taking into consideration the type, etc. Furthermore, the BRAK antagonist may be administered in the form of a pharmaceutically acceptable salt or hydrate, or may be administered together with a known pharmaceutically acceptable carrier, without limitation.

(ii) Evaluation process

The comparative evaluation target in this step includes, for example, at least one selected from the group consisting of survival rate, weight gain, hematopoietic function, and reproductive function. By comparing the evaluation targets of control non-human animals and test non-human animals administered with BRAK antagonists, it is possible to easily determine the presence or absence of side effects caused by BRAK antagonists, as well as the types of side effects and the severity of symptoms. Can be tested.

Examples of each comparative evaluation method will be explained below.

In this evaluation method, test non-human animals show no disease symptoms even after administering a BRAK antagonist, eat the same amount of food as control non-human animals in the non-administered group, and gain weight. to test whether Although control non-human animals are lighter in weight than wild-type non-human animals, they gain weight if fed a high-fat diet, so it is effective against the test non-human animals after the above administration. can be compared to.

Comparative evaluation of hematopoietic function >

In this evaluation method, the presence ratio and absolute number of various blood cells (erythrocytes, lymphocytes, neutrophils, etc.) in the peripheral blood of non-human test animals remains within the normal range even after administration of a BRAK antagonist. Verify whether it is. Control non-human animals had a reduced number of tissue maculaphages in the lungs and uterus compared to test non-human animals, but quantitative and qualitative differences were observed in blood cells in bone marrow and peripheral blood. Therefore, it can be effectively compared with control non-human animals after the above administration.

This evaluation method tests the reproductive potential of males and females over 10 weeks of age. As long as the control non-human animals have at least a genetic background that is a mixture of C57BL/6 and CBA, both males and females can produce the next generation of offspring, so the test non-human animals after the above administration can be effectively compared with

The assay method may include other steps in addition to the various steps listed above. not limited

(3) Screening method for BRAK-like mediator substances

As mentioned above, the non-human animal of the present invention lacks migration activity toward tissue macrophages due to loss of the ability to express BRAK. As a result, it has characteristics such as insulin non-resistance and a reduction in the number of macrophages in white adipose tissue. Therefore, we will attempt to screen for BRAK-like mediator substances by administering candidate substances to the non-human animals of the present invention (non-human test animals). That is, in the test non-human animal after administration, similar to the non-human animal other than the non-human animal of the present invention (control non-human animal), resistance to insulin and the increase in the number of macrophages, etc. If these characteristics are observed, it means that the patient has suffered effects similar to those caused by BRAK, even though they do not express BRAK. Therefore, the administered candidate substance can be determined to be a BRAK-like mediator substance.

That is, the present invention provides a BRAK-like mediator substance using the non-human animal of the present invention (test non-human animal) and a non-human animal other than the non-human animal of the present invention (control non-human animal). Also includes screening methods. Here, the term BRAR-like mediator substance refers to a substance that has specific migratory activity to monocytes and macrophages, and has similar activity to BRAK.

Specifically, the screening method is characterized by including the following steps (i) to (iii). Note that steps (i) and (ii) may be performed in any order, and either may be performed first or at the same time.

(i) Step of making test non-human animals and control non-human animals obese (obesity step)

(ii) Process of administering the candidate substance to the non-human test animal (administration process)

(iii) Process of comparatively evaluating the fertility status of test non-human animals and control non-human animals (evaluation process) Each of these processes will be explained below. (i) Fertilization process

The control non-human animal is an animal of the same species as the non-human animal of the present invention that is the test non-human animal, and the BRAK gene has not been disrupted (an animal in which the BRAK gene function has been lost on the chromosome). In particular, wild-type and/or BRAK hetero-deficient animals can be used. The control non-human animal is preferably an animal from the same litter as the non-human animal of the present invention (BRAK homozygous non-human animal).

Obesity in this process refers to a state in which, by feeding a non-human animal a high-fat feed, its body weight is 1.2 times or more (preferably) compared to a non-human animal of the same genotype fed a normal feed. means an increase of 1.5 times or more, more preferably 1.8 times or more, even more preferably 2 times or more). Obese mice can be obtained, for example, by feeding female mice aged 6 weeks or older a high-fat diet such as HFD32 (manufactured by Nippon Clea Co., Ltd.) for 12 weeks or more, but there are limitations. Not allowed.

When making a control non-human animal ^and a test non-human animal obese, it is preferable that the feed intake conditions be the same for both animals. .

(ϋ) Administration process .

Candidate substances to be administered to non-human test control animals (candidate substances for BRAK-like mediator substances) include various naturally occurring or artificially synthesized peptides, proteins (including antibodies), polynucleotides, and low-molecular organic compounds. can be exemplified. The candidate substance can be administered orally or parenterally, and there are no limitations, and in either case, known administration methods and conditions can be employed. The dosage can also be determined as appropriate, taking into account the type and condition of the non-human animal being tested, the type of candidate substance, etc.

(iii) Evaluation process

The comparative evaluation target in this step includes, for example, at least one selected from the group consisting of insulin resistance, the number of macrophages in white adipose tissue, and adiponectin concentration in serum. BRAK-like mediator substances can be easily screened by comparing these evaluation targets in obese test non-human animals and control non-human animals. Examples of each comparative evaluation method will be explained below.

<Comparative evaluation of insulin resistance> 'In this evaluation method, first, a test non-human animal and a control non-human animal (hereinafter referred to as each non-human animal) are subjected to a predetermined period of time (for example, 16 days in the case of mice). time) After fasting and stabilizing blood sugar levels, an insulin solution is injected intraperitoneally. Thereafter, blood sugar levels are compared over time between the test non-human animals and the control non-human animals. At this time, if there is no significant difference between the two animals, the administered candidate substance can be evaluated as the desired BRAK-like mediator substance. On the other hand, if the non-human test animal maintains a significantly lower level, it can be evaluated that the administered candidate substance is not the desired substance.

Comparative evaluation of the number of macrophages in white adipose tissue>

In this evaluation method, first, white adipose tissue from each non-human animal is collected and cut into small pieces. Next, after dissolving the fragmented tissue with various enzymes and solutions, only the macrophages are fluorescently labeled. The number of macrophages is measured using flow cytometry or a cell sorter, and the measured number is compared between the test non-human animal and the control non-human animal. At this time, if there is no significant difference between the two animals, the administered candidate substance can be evaluated to be the desired BRAK-like mediator substance. On the other hand, if the number of measurements was significantly reduced in the non-human test animals, it can be evaluated that the administered candidate substance is not the desired substance. Note that the explanation in section 2 above can be similarly applied to the fluorescent labeling of macrophages.

In this evaluation method, various known measurement kits (eg, mouse adiponectin ELISA kit (manufactured by Otsuka Pharmaceutical Co., Ltd.)) can be used. The measured adiponectin concentration in the LK serum is compared between the test non-human animal and the control non-human animal. At this time, if there is no significant difference between the two animals, the administered candidate substance can be evaluated as the desired BRAK-like mediator substance. On the other hand, if the concentration in the non-human test animal is significantly higher, it can be evaluated that the administered candidate substance is not the desired substance.

For further details of the various comparative evaluation methods exemplified above, the methods and conditions described in Examples described later can be referred to as appropriate. The screening method may include other steps in addition to the various steps described above, and is not limited. Other steps include steps including various treatments that can be carried out in known methods of screening the desired substance using knockout mice and the like. The present invention will be explained in more detail below with reference to Examples, but the present invention is not limited thereto.

[Example 1]

《Materials and methods》

1. Mice and genetic modification

The mouse BRAK gene genome sequence information (SEQ ID NO: 1) was obtained from the mouse chromosome nucleotide sequence (accession number: AC165347J) available from the public database (http: www.ncbi.nlm.nih.gov/). (Published.) Retrieved from BRAK gene base sequence (7,902 bp). In constructing the vector, as shown in Figure 1A, fragments of the homologous regions (short arm and long arm) were first prepared by PCR.

Specifically, for the short arm fragment, the spleen genomic DNA of a C57BL/6 mouse was used as a template, and the following reaction solution composition and reaction conditions were used using the following primers and rTaq polymerase (manufactured by Takara). was prepared by performing PCR.

F1 primer: 5'-TCATCTTGGTGCAGACCTCT-3, (SEQ ID NO: 2) R1 primer: 5'-CTCGGCTCGCTCCAGCTACG-3, (SEQ ID NO: 3) Reaction liquid composition >

Template (I0ng/l): 1^1

10X rTaq buffer: 2.5 μ\

2.5mM dNTP: 2μΙ

rTaq polymerase: 0.125 1

F1 primer (10 M): 0.5 μ\

R1 primer (10/iM): 0.5ΐ

Sterile water: 18.375

Total: 25^1 reaction conditions >

After heating at 95°C for 9 minutes, one cycle of "thermal denaturation, dissociation: 95°C (60sec) → ringing: 57°C (60sec) → synthesis, elongation: 72°C (60sec)" was counted. After 40 cycles, the mixture was incubated at 72°C for 7 minutes and then cooled to 4°C. On the other hand, the long arm fragment was prepared by PCR using C57BL/6 mouse spleen genomic DNA as a template, the following primers, and rTaq polymerase (manufactured by Takara) under the following reaction solution composition and reaction conditions. did.

F2 primer: 5'-TAGCATATCCTGTGGAGGAG-3 (SEQ ID NO: 4) R2 primer: 5'-AGTAGACTGAGTTCCTCTAG-3 (SEQ ID NO: 5)

Template (10ng/;ul): 1μ\

10X rTaq buffer: 2.5μ1

2.5mM dTP: 2μ\

rTaq polymerase: 0.125/il

F2 primer (10/M): 0.5 μ\

R2 Primer (ΙΟμΜ): 0.5 1

Sterile water: 18.375 ul

Total: 25 1

After heating at 95°C for 9 minutes, one cycle was ``thermal denaturation'' dissociation: 95°C (60sec) - annealing: 57°C (60sec) - synthesis 'extension: 72°C (60sec)'' for a total of 40 cycles. The cells were incubated at 72°C for 7 minutes and then cooled to 4°C. Next, the MCl-Neo gene and the diphtheria toxin (DT) gene were ligated to each fragment obtained by the above PCR to construct a targeting vector. The structure of this targeting vector basically followed the paper by Murata et al. (Murata S et al., "Growth retardation in mice lacking the proteosome activator PA28gamma", J. Biol. Chem., vol. 274, p . 38211 38215, 1999). After linearizing this vector, it was transformed into ES cell lines (TT2,

G418 was selected. A colony of proliferated ES cell clones was suspended in PBS, and using the genomic DNA prepared from the cell suspension as a template, the following primer set and rTaq polymerase (manufactured by Takara) were used. PCR was performed using the reaction solution composition and reaction conditions (PCR product size: 2,193 bp), and homologous recombinant clones were screened. <Primer set>

P1 primer:

5'-CATGCAGATCAACTGATGTCAGACACAGAC-3' (SEQ ID NO: 6) P2 primer:

' 5' CGAATGGGCTGACCGCTTCC-3' (IJ number 7) Reaction solution composition >

Template (1 colony/10 μΐ): 1 μ\

10XrTaqffl buffer: 2.5μΐ

2.5mM dNTP: 2^1

rTaq polymerase: 0.125^1

PI Primer (10/iM): 0.5^1

P2 primer (ΙΟμΜ): 0.5 μΐ

Sterile water: 18.375

Total: 25 1 reaction conditions>

After heating at 95°C for 9 minutes, ``thermal denaturation' dissociation: 95°C (60sec) → annealing: 57°C (60sec) - synthesis' elongation: 72°C (60sec)'' for a total of 45 cycles. The cells were incubated at 72°C for 7 minutes and then cooled to 4°C. Chimeric mice were obtained by microinjecting the selected ES cells into ICR mouse morulae and transplanting them into ICR mouse surrogate mothers. Wild-type, hetero-deficient, and homo-deficient mice were generated by brother-sister breeding of BRAK gene hetero-deficient Fl and F2 individuals born from mating with C57BL/6 mice, and used for experiments.

For genotyping, tail fragments were incubated at 56°C in DNA extraction buffer [100 mM Tris HCl pH 7.5, 300 mM KCl, 0.5% NP40, proteinase K (10 μg/ml)] at 50°C. Insulated. This treatment solution was heated at 98°C for 20 minutes, and one set of the following two types of primers (wild type and BRAK knockout) was added using Ιμΐ as a template. PCR was performed using the following reaction solution composition and reaction conditions using the following reaction solution composition and reaction conditions: Primer set>

•One set of primers for wild type (PCR product size: 210 bp)

F3 primer: 5'-AGACCTGTACGGCGGCGACTC-3' (SEQ ID NO: 8) R3 primer: 5'-GTCCGATCTAACCCTAGGTTG-3, (SEQ ID NO: 9)

Primer set for BRAK knockout type (PCR product size: 251 bp) F3' primer: 5'-CTGCATCTGCGTGTTCGAAT-3, (SEQ ID NO: 10) R3 primer: 5'-GTCCGATCTAACCCTAGGTTG-3, (SEQ ID NO: 9) Reaction liquid composition>

Template (tail fragment /50 1): ΙμΙ

10X rTaq buffer: 2.5μΙ

2.5mM dP: 2μΙ

rTaq polymerase: 0.125μ1

F3 or F3' primer (ΙΟ, Μ): 0.5^1

R3 primer (ΙΟμΜ): 0.5 μΐ

Sterile water: 18.375/l

Total: 25μ1 reaction conditions>

After heating at 95°C for 9 minutes, one cycle was ``thermal denaturation, dissociation: 95°C (60sec) - annealing: 57°C (60sec) - synthesis and extension: 72°C (60sec)'' for a total of 40 cycles. The cells were incubated at 72°C for 7 minutes and then cooled to 4°C.

2. Southern blot and Northern blot analysis

Extract high-molecular genomic DNA and total RNA using standard methods, and transfer to a nylon membrane. (Hybond-N, manufactured by Amersham). A 32p-dCTP-labeled probe was used for hybridization, and radiation signals were detected using BAS3000 (manufactured by Fuji Film).

Mouse BRAK genomic DNA fragment (390 bp) was used for genomic Southern blot analysis of BRAK. This fragment was prepared by PCR using the spleen genomic DNA of a C57BL/6 mouse as a template and using the following primers and rTaq polymerase (manufactured by Takara) under the following reaction solution composition and reaction conditions.

F4 primer: 5'-GCTTCCAGATGTGAGATCCAG-3' (SEQ ID NO: 1 1) R2 primer: 5'-AGTAGACTGAGTTCCTCTAG-3' (SEQ ID NO: 5)

Template (10ng/l): Ι μ Ι

10X buffer for rTaq: 2.5^1

2.5mM dNTP: 2 μ\

rTaq polymerase: 0.125 μΐ

F4 Primer (ΙΟ μ Μ): 0.5 μ 1

R2 primer (ΙΟ μ Μ): 0.5 μ 1

Sterile water: 18.375 1

tl 25 μΐ reaction conditions〉

After heating at 95°C for 9 minutes, one cycle of ``thermal denaturation, dissociation: 95°C (60sec) → a-21 ring: 57°C (60sec) → synthesis, elongation: 72°C (60sec)'' was counted. 40 cycles were performed, and the mixture was incubated at 72°C for 7 minutes and then cooled to 4°C. On the other hand, open reading frame fragments of mouse BRAK cDNA, β-actin, and β-tubulin cDNA were used for Northern blot analysis. 3. Insulin resistance and glucose tolerance test

After the mice were fasted for 16 hours, insulin (manufactured by Wako Pure Chemical Industries, Ltd.) (0.75 mU/g) or glucose (manufactured by Wako Pure Chemical Industries, Ltd.) (0.75 mg/g) was intraperitoneally injected. Blood sugar levels after a certain period of time were measured over time using Daltest Ace R (manufactured by Sanwa Kagaku Kenkyusho). High-fat diet feeding was carried out on HFD32 (manufactured by Nippon Clea Co., Ltd.) for 12 weeks or more in female mice aged 6 weeks or older.

4. ELISA assay and blood lipid analysis

To measure the concentrations of insulin, levtin, and adiponectin in serum, use the Levis Mouse Insulin Kit (manufactured by Shibayagi Co., Ltd.), the Mouse Levutin Measurement Kit (manufactured by Morinaga Institute of Biosciences), and the Mouse Adiponectin ELISA Kit (Otsuka Pharmaceutical Co., Ltd.), respectively. (manufactured by). In addition, the concentrations of cholesterol, triglyceride, and free fatty acids in serum were measured using Free Cholesterol E-Test, Triglyceride E-Test, and NEFA C-Test, manufactured by Wako Pure Chemical Industries, Ltd., respectively. Ta. Statistical processing was performed using Excel (manufactured by Microsoft), and significance testing was performed using StatView-J5.0 (manufactured by Abacus).

5. Macrophage entrapment and flow cytometry

After collecting the white adipose tissue around the uterus and cutting it into small pieces with scissors, it was placed in 10 mL of collagenase (1 mg/ml)/0.8% Dispase (Roche) solution per lg at 37 degrees for 45 minutes. , shake slowly. Undissociated tissue pieces were removed using a 70 cell strainer (manufactured by Falcon) and centrifuged at 1200 rpm for 5 minutes. The red blood cells in the precipitated cells were hemolyzed with 0.1M NH ₄ CM7mM Tris HCl pH 7.2, and then placed in PBS containing 5% fetal calf serum (FCS) and mouse IgG (manufactured by Sigma) (10/g/ml). The cells were then reacted with pyotinylated anti-Macl antibody (Ml/70, manufactured by BD Pharmingen) (10 g/ml) for 30 minutes on ice. Add 20 times the volume of ice-cold 5% FCS-PBS and centrifuge, then add FITC-labeled streptavidin (manufactured by BD Pharmingen) (10 / g/ml) in 5% FCS-PBS on ice for 30 minutes. Made it react. After adding 20 times the volume of ice-cold 5% FCS-PBS and centrifuging, The cells were suspended in PBS (2 μg/ml) containing Propidium iodide (manufactured by Sigma), and the fluorescence intensity of the cells was measured using FACS Aria (BD). The content ratio of Macl-negative and Macl-positive cells was calculated using FACS DiVa software (BD). 6. Tissue immunostaining

The white adipose tissue surrounding the uterus was fixed with 4% paraformaldehyde/PBS and then embedded in Noraffin. The sections (6 μιη thickness) were reacted with biotinylated anti-F4/80 antibody (ΒΜ8, manufactured by Caltag) (10 μg/ml) for 30 minutes at room temperature. After washing with PBS, it was reacted with streptavidin labeled with horseradish peroxidase (manufactured by BD Pharmingien Co., Ltd.) (10 g/ml) for 30 minutes at room temperature, and then reacted with diaminobenzidine as a substrate. Colored. For details of the color development method, we referred to the description in a paper previously reported by the present inventors (Hara T et al., Distinct roles of oncostatin M and leukemia inhibitory factor in the development of primordial germ cells and Sertoli cells in mice, Dev. Biol., vol. 201, p. 144:153, 1998).

《Experiment history and results》

1. Establishment of BRAK gene-disrupted mouse line

By crossing chimeric mice with a high contribution rate of ES cell clones that had undergone homologous recombination at the mouse BRAK gene locus, mice with heterozygous BRAK gene deletion were obtained. The genotypes of mice born by mating between heterozygous mice were: wild type (+/+): heterozygotic (+/_): homozygotic (-) = 69: 168: 52. Southern plot analysis of spleen genomic DNA from (+Λ), (+/-), (_/ -) mice confirmed that a Neo cassette was inserted into the translation initiation site of the BRAK gene ( Figure 1B). Mouse BRAK mRNA is mainly expressed in the brain, lungs, and skeletal muscles, but the expression level was halved in (+/_) mice, and no mRNA was detected in (_/-) mice (Fig. 1C). The above results showed that BRAK gene expression was completely disrupted in BRAK (-/-) mice as planned.

2. Characteristics of BRAK gene-disrupted mice The male:female ratio of BRAK (-/-) mice at birth was approximately 3:5, and both sexes were fertile with no apparent abnormalities observed. No significant differences were observed in the composition of blood cells in peripheral blood, spleen, and bone marrow. However, male (_/-) mice over 6 months of age weigh less than (+/+) or (+/-) male mice from the same litter and have smaller white adipose tissue in the abdominal cavity. was often observed (Figure 2A). Even in 6-month-old adult female mice, the average body weight of the (-/-) mouse group was 16.5% smaller than the (+/+) or (+/-) mouse group (Figure 2C), and the (-/-) mouse group The weight-to-body weight ratio of periuterine white adipose tissue was reduced to 45% of that in the control mouse group (Fig. 2E). The average food intake per body weight in the (-/_) female mouse group was slightly higher (12.7%) than in the (+/+) (+/-) female mouse group (Figure 2D). , there was no difference between the two groups (Figure 2E). When paraffin sections of white adipose tissue were observed, the size of the adipocytes constituting the white adipose tissue of (-/_) mice was almost the same as that of (V-) mice (see A in Figure 3). and B). Furthermore, there was no significant difference in the number of F4/80-positive macrophages per fat area between the two groups. The total number of Macl (monocyte/macrophage marker) positive cells per individual, which are dissociated from the white fat surrounding the cubs by enzymatic treatment, was calculated based on fax data, and it was found that the (- /_) mouse group was Macl positive. The number of cells was 35.3% lower than that in the (+/+) (+/_) mouse group (Figure 2F).

Since we suspected that BRAK homozygous-deficient mice had a reduced ability to accumulate fat in the body, we next induced experimental obesity by feeding BRAK (-/-) female mice and control littermate mice a high-fat diet. . When body weight was monitored over a 14-week period, the (- /-) mouse group also gained weight over time, but the average weight of the (+/+) (+/-) mouse group was always 20% higher. (Figures 2B, 2C). The high-fat food intake per body weight of (-/_) mice was the same as that of (+/+) (+/_) mice (Figure 2 D), indicating that the difference in body weight was not due to appetite. It is inferred. Next, when we compared the weight ratio of periuterine white adipose tissue per body weight using mice fed a high-fat diet, there was no significant difference between the (-/_) mouse group and the control mouse group. . Histological findings also confirmed that individual adipocytes in the white adipose tissue of (-/_) mice were enlarged compared to the normal diet group, but were almost the same as the adipocytes of (+/_) mice. (Figure 3C and D). However, in the white adipose tissue of obese (+/-) mice, there are more F4/80-positive macrophages than in mice. It surrounded adipocytes (Figure 3C). Consistent with this result, the total number of Mac l-positive cells per individual dissociated from the periuterine white fat of obese mice by enzymatic treatment was 3.8 in (+/+) (+/_) mice than in the normal diet group. However, the increase was only slight in the (- /_) mouse group (Figure 2F). These results are consistent with the fact that BRAK is a chemokine that acts on activated macrophages, and that BRAK mRNA expression in white adipose tissue increases markedly with obesity (described below). Interestingly, in the (+/+) (+/-) mouse group that became obese due to high-fat diet feeding, the number of Mac I negative cells also increased 8.6 times compared to when fed a normal diet (Fig. 2F). This result suggests that BRAK is also involved in the accumulation of non-hematopoietic cells present in obese adipose tissue. There was no difference in the ratio of white fat to body weight between the two mouse groups, but the liver in the (+/+) (+/_) mouse group was extremely enlarged and fatty liver was observed (Figure 2E) (average 3.7 g, n=6). In contrast, the livers of the (-/_) mouse group were approximately the same in color and weight (average 1.6 g, n=5) as the livers of normal mice. The kidney weights of mice fed a high-fat diet did not differ between the (_/-) and (+/+) (+/_) mouse groups, and were similar to the kidney weights of mice fed a normal diet. (Figure 2E). The above results indicate that BRAK homozygous-deficient mice are able to newly produce white fat when fed a high-fat diet, but macrophage recruitment to it is markedly inhibited, and furthermore, lipid deposition within the liver parenchyma is reduced. This was to show that it would not happen. 3. Glucose metabolic ability and insulin response of BRAK gene-disrupted mice

Accumulation of white adipose tissue and fatty liver in obese mice cause abnormalities in the individual's blood sugar level regulation. (+/+) (+/-) The post-fasting blood sugar levels of female mice were the same as those of (_/-) female mice when fed a normal diet, but increased in the group fed a high-fat diet. (Figure 4A). Even in female mice fed a high-fat diet (- /_), blood sugar levels were elevated compared to those fed a normal diet. Next, we investigated the blood insulin concentration in the absence of dietary restrictions, and found that (- /_) female mice fed a normal diet had a lower concentration of insulin than (+/+) (+/-) female mice. % (Figure 5A). When the mice were made obese by feeding them a high-fat diet, the value increased 12.7 times in the (+/+) (+/_) female mouse group and 9.81 times in the (-/_) female mouse group. The blood insulin concentration of the latter was 32.4% of that of the former, which was a relatively low level (Fig. 5A). To find out whether BRAK homozygous mice have a defect in insulin responsiveness, we injected insulin into a group of mice raised on a high-fat diet and measured changes in blood sugar levels over time. In the (+/+) (+/-) mouse group, the drop in blood sugar level slowed and became insulin resistant, but in the (- /-) mouse group, the (+/+) (+/_) group was fed a normal diet. An insulin response almost equivalent to that in the mouse group was observed (Figure 4B). Next, when we examined the increase in blood sugar levels caused by glucose injection, we found that even the (-/-) mouse group raised on a high-fat diet had decreased glucose tolerance; This occurred more acutely than in the (+/+) (+/-) mouse group (Figure 4C).

The (-/-) mouse group fed a normal diet was not significantly different from the (+/+) (+/-) mouse group in both the insulin resistance test and the glucose tolerance test (Figures 4B and 4C). ). The above results indicate that the ability of BRAK homozygous-deficient mice to regulate blood sugar by insulin is normal, and they maintain high insulin responsiveness even when they are made obese by being fed a high-fat diet (i.e., they do not show insulin resistance). This was an indication that The cause of decreased glucose tolerance in the obese (-/-) mouse group is still unknown, but it may be due to decreased insulin secretion.

4. Adibokine and lipid production in BRAK gene-disrupted mice

Levutin and adiponectin secreted from mature adipocytes are already known to play important roles in obesity and blood sugar regulation. Therefore, we compared the blood concentrations of these adivocaine between the (-/-) and (+/+) (+/-) mouse groups. First, the leptin concentration is almost positively correlated with the average white fat weight of each mouse group, and the blood concentration in the (_/-) mouse group fed a normal diet was (+/+) (+/_ ) group was 54.0% (Figure 5B). On the other hand, in mice fed a high-fat diet, blood levutin concentrations increased to the same level in both the (-/_) and (+/+) (+/_) groups (Figure 5B). However, when we calculated the levtin production rate per weight of white fat, it was 19.9% higher in the normal diet group and 26.2% higher in the high fat group in the (_/-) mouse group. Therefore, it is inferred that BRAK homozygous deficiency slightly increases levtin production in white fat. Next, we compared the blood concentration of adiponectin between the (-/_) mouse group and the (+/+) (+/_) mouse group, and found that (- /_) Lower level in mouse group (Figure 5C). When the adiponectin production rate per weight of white fat was calculated, it was 3.1 times higher in the (-/-) mouse group than in the normal diet group and 2.4 times higher in the high fat group. Adiponectin is an important secreted protein that improves insulin resistance that occurs with obesity, and our results suggest that BRAK negatively regulates the blood concentration of adiponectin in individuals.

Finally, we measured the blood lipid concentration, which is a barometer for metabolic syndrome. In the high-fat diet-fed (-/-) mouse group, blood cholesterol levels were 21.5% lower than in the high-fat diet-fed (+/+) (+/-) group (Figure 5D). There were no significant differences in the concentrations of triglycerides and free fatty acids, which are representative of fats (Figures 5E and 5F).

5. BRAK expression in white adipose tissue

As mentioned above, differences in the development and maturation of white adipose tissue were observed in BRAK homozygous-deficient mice, so we analyzed the expression of BRAK mRNA in various organs of the mice. As previously known, BRAK mRNA was synthesized at high levels in the brain, lung, ovary, and skeletal muscle, and at low levels in the bone marrow and kidney (Figure 6A). The present invention showed that BRAK mRNA was also expressed in the uterus, white adipose tissue, and brown adipose tissue (Fig. 6A). More interestingly, the expression level of BRAK mRNA increased approximately 5-fold in the white adipose tissue of (+/-) mice fed a high-fat diet and made obese (Figure 6B). In the obese (+/-) mouse group, TNF mRNA expression was also increased. Therefore, BRAK is considered to be a new member of adipokine genes whose transcription is induced with obesity.

《Consideration》

In the BRAK knockout mice of the present invention, the increase in liver weight and fattening that are observed in wild-type mice when raised on a high-fat diet do not occur, and the number of macrophages inside the white adipose tissue is significantly lower than in wild-type mice. Ta. Furthermore, obesity-associated insulin resistance was not observed in BRAK knockout mice. Furthermore, BRAK mRNA expression was found to be significantly elevated in obese adipose tissue. These experimental facts indicate that BRAK is specifically involved in the induction of insulin resistance due to obesity. This indicated that it was a chemokine.

BRAK is known as a chemokine that specifically attracts macrophages activated by _PGE2 (Kurth I et al., J. Exp. Med., vol. 194, p. 855-861, 2001). . In mice that have consumed excessive amounts of food, it is presumed that activated macrophages in the surrounding tissues migrate toward 'BRAK secreted from white adipose tissue. Macrophages infiltrating adipose tissue are thought to secrete mediators such as TNF and another macrophage migration factor, MCP-1, which induce insulin resistance in adipocytes and the liver.

In BRAK knockout mice, adiponectin production from white adipose tissue was higher than in control mice. Adiponectin acts on the liver and muscles to increase fatty acid combustion through activation of PPAR (Yamauchi T et al., Nat. Med., vol. 8, p. 1288-1295, 2002). Yamauchi et al. reported that when adiponectin was administered to genetically obese mice KKAy, obesity was not improved, but fat content was reduced and insulin resistance was improved (Yamauchi T et al., Nat. Med., vol. 7, p. 941-946, 2001). The reason that the BRAK (-/-) mouse group did not exhibit fatty liver symptoms even when fed a high-fat diet is thought to be due to the action of adiponectin. The phenotype of this mouse contradicts the previously proposed independence of adipose tissue hypertrophy and the acquisition of insulin resistance, and further suggests that insulin resistance is caused by a BRAK-mediated tissue macrophage. This indicates that oral phage accumulation is essential. Obesity-related disease model mice that have been reported so far (Understanding Experimental Medicine Series: Understanding Lifestyle-related Diseases, edited by Masato Kasuga, Yodosha, 2005) include lebutin-deficient mutant mouse, nuclear factor peroxisome proliferator-activated receptor Mutant mice, such as delta (PPAR δ)-deficient mice and Angiopoietin-related growth factor (AGF)-deficient mice, whose food intake exceeds their energy metabolism, resulting in extreme obesity and insulin resistance. There are many examples of genetically modified mice. Conversely, mice that are resistant to obesity have recently been reported, including double-deficient fatty acid binding protein aP2/mall mice and AGF transgenic mice. In these genetically modified mice, white adipose tissue hypertrophy does not occur even when raised on a high-fat diet due to enhanced energy metabolism, and both obesity and insulin resistance are improved. Genetically modified mice that become obese but have improved insulin resistance include:

JNK gene-deficient mice have been reported (Hirosumi J et al., Nature, vol. 420, p. 333-336, 2002). The BRAK knockout mouse of the present invention is the first case in which insulin resistance due to obesity has been improved among mice genetically modified with chemokines and cytokines that act on macrophages. In this example, it was found that BRAK is essential for the acquisition of insulin resistance due to obesity. The findings obtained through the present invention indicate that BRAK antagonists are useful as therapeutic agents for a new type of type 2 diabetes, and this knowledge also makes it possible to administer BRAK antagonists to patients with type 2 diabetes. Can be used to prevent or treat type 2 diabetes.

Furthermore, the efficacy and side effects of the above therapeutic agent can be easily tested by comparing it with the BRAK knockout mice of the present invention (non-administered group).

Recently, it has been shown that BRAK is involved in the migration and differentiation of dendritic progenitor cells and the invasion of cancer cells. BRAK is a molecule originally discovered as a CXC chemokine whose expression is lost in cancer cells, and it is clear that BRAK knockout mice will be useful for research into cancer immunity and metastasis mechanisms. If the BRAK knockout mouse is sold, researchers around the world who are interested in macrophage organ regeneration and cancer will likely obtain this mouse and use it to approach the physiological functions of BRAK. It is expected that the scope of use of this mouse will continue to expand as research progresses.

[Example 2]

《Materials and methods》

1. Generation of BRAK transgenic mice

Information on the cDNA base sequence (SEQ ID NO: 12) encoding mouse BRAK was obtained from a known document (Sleeman MA et al., Int. Immunol., vol. 12, p. 677-689, 2000). In addition, this nucleotide sequence information (SEQ ID NO: 12) has been published in the public database (http://www.ncbi.nlm.nih.gov/) as “Accession number: AF144754J.” , BMAC (B cell- and monocyte-activating chemokine) described in this document corresponds to BRAK mentioned in this specification. It is.

Of the above cDNA base sequence (SEQ ID NO: 12), the open reading frame (300 bp from the 203rd to the 502nd (from the start codon ATG to the end codon TAG); SEQ ID NO: 13 is the amino acid sequence after translation. ) was cloned. Specifically, total RNA from a C2C12 myoblast cell line obtained from a public cell bank was used as a template, and single-stranded cDNA was generated using the Superscript First strand cDNA synthesis kit and random hexamer (manufactured by Invitrogen). After synthesizing, prepare RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction) using the following reaction solution composition and reaction conditions, including the following primer set.

F5 primer: 5'-ATGAGGCTCCTGGCGGCCGCG-3' (SEQ ID NO: 14) R5 primer: 5'-CTATTCTTCGTAGACCCTGCG-3' (SEQ ID NO: 15) <Reaction solution composition>

cDNA template (40ng/il): 1μ\

buffer for lOXrTaq: 2.5^1

2.5mM dTP: 2μΙ

rTaq polymerase: 0.125^1

F5 primer (lO uM): 0.5 μΐ

R5 primer (ΙΟ Μ): 0.5 μ ΐ

Sterile water: 18.375

Total: 25/l

After heating at 95°C for 9 minutes, one cycle was ``thermal denaturation/dissociation: 95°C (60sec) → annealing: 57°C (60sec) → synthesis and elongation: 72°C (60sec)'' for a total of 40 cycles. The cells were incubated at 72°C for 7 minutes and then cooled to 4°C. Next, the DNA fragment obtained by the above RT-PCR was transformed into DNA consisting of the promoter-enhancer region derived from the mouse creatine kinase (MCK) gene (6.5kb; Cox GA et al., Nature, vol. 364, p. 725-729 , 1993), and a PolyA signal sequence derived from the SV40 virus (pcDNA3.1 vector, Invitrogen) was further ligated to the 3' end of the BRAK cDNA fragment. The DNA fragment thus obtained was purified by electrophoresis.

The purified DNA fragment was microinjected into the pronuclei of C57BL/6 mouse fertilized eggs. DNA-injected eggs were transplanted into the uterus of pseudo-pregnant mice, and among the offspring mice, those with the desired DNA fragment inserted were selected and used as the first-generation MCK-BRAK transgenic mice. .

Mice with the desired DNA fragment inserted were selected by PCR (genotyping) of tail DNA. Specifically, first, the tail fragment of the obtained mouse pup was added to 50 μl of DNA extraction buffer (100 mM Tris HCl pH 7.5, 300 mM KCl, 0.5% NP40, proteinase K (10 /i g/ml)). Insulated overnight at 56°C. This treatment solution was heated at 98°C for 20 minutes, and then PCR was performed using IΙ and N as templates, the following set of primers, and rTaq polymerase (manufactured by Takara) under the following reaction solution composition and reaction conditions. went.

· For MCK-BRAK transgenic mice (PCR product size: 430 bp)

F6 primer:

5'-CAGGATGTGGCCACATCAGGCAACTTGGGC-3' (SEQ ID NO: 16) R6 primer:

5'-CCAGGCATTGTACCACTTGA-3' (SEQ ID NO: 17) Reaction liquid composition >

Template (tail fragment /50 1): 1 ^ 1

buffer for lO X rTaq: 2.5 1

2.5mM dNTP: 2 μ\

rTaq polymerase: 0. 125 1

F6 primer (10; u M): 0.5 μ 1

R6 primer (ΙΟ μ Μ): 0.5 μ ΐ

Sterile water: 18.375 1

Total: 25 1 reaction conditions>

After heating at 95°C for 9 minutes, ``thermal denaturation'' dissociation: 95°C (60sec) → annealing: 57°C (60sec) → synthesis ``elongation: 72°C (60sec)'' for a total of 45 cycles. The cells were incubated at 72°C for 7 minutes and then cooled to 4°C.

2. Western blot analysis

C2C 12 myoblast cell line obtained from a public cell bank was cultured in Dulbecco's modified Eagle's medium (DMEM) containing 5% horse serum for 4 days to induce muscle differentiation. After culturing in serum-free DMEM medium for 16 hours, myocytes were treated with 100 nM mouse BRAK (R&D Systems) for 1 hour, and further stimulated with 10 nM insulin for 10 minutes at 37°C. did. As a control group, cells not treated with BRAK and cells to which neither BRAK nor insulin was added were used. These cells were lysed, and 20 g of the protein mixture was subjected to SDS-polyacrylamide electrophoresis and Western blot analysis using Akt-pSer473 antibody and anti-Akt antibody (Cell Signaling).

Akt is a serine/threonine kinase also called protein kinase B (PKB), which is activated through the PI3 kinase pathway and is involved in various phenomena such as insulin metabolism. "result"

In order to clarify whether the improvement in insulin resistance caused by a high-fat diet observed in BRAK-knockout mice (see Example 1) was due to the BRAK deficiency itself or to an indirect effect, As mentioned above, we created transgenic mice (MCK-BRAK transgenic mice) in which BRAK was forced to specifically express in skeletal muscle (Fig. 7A) and crossed them with BRAK knockout mice. An insulin resistance test showed that BRAK knockout mice fed high fat and MCK-BRAK transgenic mice lost the insulin sensitivity that BRAK knockout mice fed high fat had, whereas BRAK hetero knockout mice fed high fat lost the insulin sensitivity of BRAK knockout mice fed high fat. Insulin resistance similar to that in mice was observed (Figure 7B). On the other hand, no insulin resistance was observed in BRAK knockout'MCK-BRAK transgenic mice fed a normal diet (Fig. 7C). Based on the above, recovery of insulin resistance due to forced expression of BRAK is considered to be a phenomenon that occurs only in individuals that have become obese due to high-fat feeding.

Next, in order to examine the possibility that BRAK directly induces insulin resistance, differentiated C2C12 myocytes were stimulated with insulin in the presence or absence of BRAK, and insulin induced the 473rd stage of Akt. The effect on phosphorylation (activation state) of the serine residue was analyzed. As a result, BRAK partially inhibited insulin-stimulated Akt phosphorylation (Figure ^7D ). When the density of the band after Western blotting was quantified, the Akt phosphorylation rate was reduced to 59.2% compared to insulin stimulation alone ₍

《Consideration》

The experimental results showed that insulin resistance was restored in high-fat-fed BRAK knockout mice by mating with MCK-BRAK transgenic mice, indicating that BRAK is a direct causative agent for the development of insulin resistance in obese mice. It shows. Furthermore, although BRAK produced by skeletal muscle may circulate throughout the body through the bloodstream, it is unlikely that it increases local BRAK levels in visceral white adipose tissue. Preliminary experimental results indicate that the number of mucophages in the white adipose tissue of BRAK knockout-MCK-BRAK transgenic mice fed with high fat The number of macrophages in the white adipose tissue of high-fat-fed BRAK heteroknockout mice decreased to approximately 44%. Although this result does not indicate that macrophage infiltration induction by BRAK in obese adipose tissue does not contribute to the development of insulin resistance, it does indicate that BRAK has a different type of glucose metabolism regulatory activity than macrophage recruitment. This strongly indicates that This prediction was supported by experimental results showing that BRAK directly inhibits insulin signaling to C2C12 muscle cells. BRAK-knockout' MCK-BRAK transgenic mice when fed a normal diet did not show systemic insulin resistance, suggesting that the insulin signal-inhibiting effect of BRAK alone is coordinated with some other obesity factor. It is thought that this mechanism acts as a mechanism to cause the development of systemic insulin resistance. Industrial applicability

According to the present invention, there is provided a knockout non-human animal in which the BRAK gene has been disrupted, ie, a non-human animal in which the ability to express BRAK/CXCL14 has been lost. The non-human animal of the present invention is intended to elucidate the relationship between BARK and the chemotaxis of monocytes and macrophages to white adipose tissue, and furthermore to clarify the relationship between BARK and type 2 diabetes, which is mainly caused by insulin resistance. It is extremely useful for elucidating the

The present invention also provides a method for screening BRAK antagonists. The screening method of the present invention uses the non-human animal of the present invention that can survive for a long period of time and a control non-human animal (both made obese) to determine whether the candidate substance is an antagonist of the BRAK receptor. It is extremely useful because it allows you to easily evaluate whether the Furthermore, the present invention provides a method for testing the side effects of a BRAK antagonist using the non-human animal of the present invention and a control non-human animal.

The present invention is very useful for developing drugs for type 2 diabetes. Sequence listing free text

SEQ ID NO: 2: Synthetic DNA

SEQ ID NO: 3: Synthetic DNA

SEQ ID NO: 4: Synthetic DNA SEQ ID NO:5: Synthetic DNA SEQ ID NO:6: Synthetic DNA SEQ ID NO:7: :Synthetic DNA SEQ ID NO:8: :Synthetic DNA SEQ ID NO:9: :Synthetic DNA SEQ ID NO:10: :Synthetic DNA SEQ ID NO:1 1: :Synthetic DNA SEQ ID NO. 14: Synthetic DNA Sequence Number 15: Synthetic DNA Sequence Number 16: Synthetic DNA Sequence Number 17: Synthetic DNA

Claims

1. Knockout non-human animal with disrupted BRAK gene.

2. The non-human animal according to claim 1, which does not exhibit insulin resistance when made obese.

3. The non-human animal according to claim 1 or 2, wherein the number of magmouth phages in white adipose tissue is reduced when the animal is made obese.

'

4. The word according to any one of claims 1 to 3, wherein the non-human animal is a rodent.

Non-human animals.

105. The non-human animal according to claim 4, wherein the rodent is a mouse.

6. A method of screening BRAK antagonists,

Surroundings

The non-human animal according to any one of claims 1 to 5 is used as a control non-human animal, and a non-human animal other than the control non-human animal is used as a test non-human animal,

making the control non-human animal and the test non-human animal obese;

15 the step of administering the candidate substance to the non-human test animal, and

The method described above, comprising the step of comparatively evaluating the obesity status of the control non-human animal and the test non-human animal.

7. A method for assaying side effects of BRAK antagonist, comprising:

A step of administering a BRAK antagonist to the non-human test animal, and a step of comparatively evaluating the control non-human animal and the non-human test animal.

The method comprising: