JP5246740B2 - Proteins and receptors thereof that change in expression in muscle cells in response to exercise, genes encoding them, and screening methods using them - Google Patents

Description

  The present invention relates to a method for measuring the expression level of a gene and detecting the intensity of a motion stimulus loaded on a muscle based on the variation thereof, a measurement kit used for the method, and various uses of the gene or gene product.

Moderate exercise is extremely important in maintaining normal metabolism in the muscle. In particular, it is becoming clear that exercise therapy, together with diet therapy, exerts a great effect as prevention and treatment of diabetes, which has become a global social problem in recent years.

On the other hand, even if it is considered that exercise therapy is effective due to the characteristics of these diseases, there are many cases where exercise therapy cannot be effectively used depending on the individual's pathological condition and lifestyle.

In addition to exerting good effects, exercise also has bad aspects such as active oxygen production and inflammatory action, and it is necessary to consider proper exercise (exercise quality) and moderate intensity (exercise intensity) There is. In addition, it is important to exclude the bad aspect of exercise in order to bring about a good effect of exercise more effectively.

Furthermore, “moderate exercise” and “moderate intensity” during normal or pathological conditions vary widely among individuals, and what indicators should be evaluated are based on empirical judgment. There is no large, unified view based on scientific evidence.

Currently, there is a method for evaluating the exercise effect by collecting a small amount of muscle before and after exercise and measuring the sugar / lipid metabolic state as one of the means considered to be effective for such exercise evaluation (Non-patent Document 1). ), Because it is a highly invasive method and the load on the subject is too great, and it is difficult to use as a general evaluation method because the evaluation method is complicated and requires skill. Not reached.

In addition, when conducting basic research aimed at analyzing how exercise effects affect muscles, exercise can be performed by applying electrical pulse stimulation to muscles collected from animals or cultured muscle cells. Although attempts have been made (Non-Patent Document 2), there are also methods for simply evaluating the intensity of the exercise and methods for simply evaluating the effect on the metabolic environment on muscles. Not established.
Shenk S and Horowitz JF (2007) Acute exercise increases triglyceride synthesis in skeletal muscle and prevents fatty acid-induced insulin resistance. J Clin Invest 117 (6): 1690-8 Brevet A, Pinto E, Peacock J, Stockdale FE (1976) Myosin synthesis increased by electrical stimulation of skeletal muscle cell cultures. Science 193 (4258): 1152-1154 Nedachi T and Kanzaki M, Am J Physiol Endocrinol Metab, 2006, 291 (4), E817-828

  Accordingly, an object of the present invention is to solve the above problems. That is, more specifically, the object of the present invention is to use an enzyme protein, a secreted protein, or a receptor thereof whose expression level varies in relation to exercise intensity using muscle or cultured muscle cell line collected from an animal. A minimally invasive and simple method for measuring the effects of exercise on normal or pathological conditions using the body or genes encoding them on the overall metabolic state, including assessment of exercise intensity and muscle energy consumption Such as providing a method.

  The inventors of the present invention have so far constructed a special cultured cell system capable of observing a pseudo motor stimulus by applying an electric pulse stimulus to the cultured muscle cell system (Japanese Patent Laid-Open Nos. 2006-296282 and 2006). No. 340637). The present inventor has further developed this original research and developed a method for rough purification of myotube cells and a method for comprehensive gene expression analysis. The present inventors have found that the gene to be encoded changes in expression in an exercise-dependent manner, and further, identified genes / proteins that regulate or link muscle health and metabolism, and completed the present invention.

That is, the present invention relates to the following aspects.
[Aspect 1] A method of measuring the expression level of at least one gene selected from the following gene group consisting of 54 types of genes and detecting the intensity of the exercise stimulus loaded on the muscle based on the variation:
(1) CXC ligand 1, (2) CXC receptor 7 (CXCR7), (3) CXC ligand 5, (4) Oncostatin R, (5) Wnt inhibitory factor-1 (WIF-1), (6) GPR43, ( 7) VEGF-D, (8) SCF / kit-ligand, (9) COX2, (10) prrx1, (11) HDAC9, (12) Interleukin-6 (IL-6), (13) FKBP11, (14) oxysterol binding protein-like 1 (OSBPL1), (15) SERPING, (16) Ubiquitin-conjugating enzyme E2B, (17) TBC domain family member1, (18) Ubiquitin-conjugating enzyme E2N, (19) Fibroblast growth factor 21, ( 20) Adaptor-related protein complex 2, (21) Ubiquitin-conjugating enzyme E2 variant 2, (22) IGFBP5, (23) Ubiquitin specific peptidase 15, (24) CD28 antigen, (25) Epsin 2, (26) Toll interacting protein, (27) von Hippel-Lindau syndrome homolog, (28) Ubiquitin specific peptidase 12, (29) SUMO / sentrin specific peptidase 2, (30) Heme oxygenase (decycling) 1, (31) Dual specificity phosphatases 18, (32 ) Ubiquitin specific peptidase 22, (33) Ubiquitin protein ligase E3A, (34) Protein phosphatase 1, (35) Hect domain and RCC1-like domain , (36) Calpastatin, (37) RAB GTPase activating protein 1, (38) Ubiquitin-conjugating enzyme E2L3, (39) interleukin 13 receptor alpha1, (40) RAD23a homolog (S. cerevisiae), (41) Ubiquitin specific peptidase 9 (42) Ubiquitin fusion degradation 1 like, (43) protein phosphatases 4 catalytic subunit, (44) ubiquitin-conjugating enzyme E2D3, (45) IGFBP6, (46) Sphingosine phosphate lyase 1, (47) adapter protein complex AP-2 alpha2 subunit, (48) RAB10, (49) HECT domain containing 1, (50) Ubiquitin specific peptidase 14, (51) Ubiquitin specific peptidase 32, (52) interleukin-1 receptor associated kinase 2, (53) ubiquitin-conjugating enzyme E2M, (52) adapter-related protein complex 3, (53) Ubiquitin specific peptidase 4, and (54) Dual specificity phosphatases 4.
[Aspect 2] A method of measuring the expression level of at least one gene selected from the gene group consisting of the following 15 genes and detecting the intensity of the exercise stimulus loaded on the muscle based on the enhancement amount:
(1) CXC ligand 1, (2) CXC receptor 7 (CXCR7), (3) CXC ligand 5, (4) Oncostatin R, (5) Wnt inhibitory factor-1 (WIF-1), (6) GPR43, ( 7) VEGF-D, (8) SCF / kit-ligand, (9) COX2, (10) prrx1, (11) HDAC9, (12) Interleukin-6 (IL-6), (13) FKBP11, (14) oxysterol binding protein-like 1 (OSBPL1), and (15) SERPING.
[Aspect 3] The method according to Aspect 2, wherein the gene group consists of CXC ligand 1 and CXC ligand 5.
[Aspect 4] A method for measuring the expression level of at least one gene selected from the gene group consisting of the following 39 genes and detecting the intensity of the exercise stimulus loaded on the muscle based on the amount of attenuation:
(16) Ubiquitin-conjugating enzyme E2B, (17) TBC domain family member1, (18) Ubiquitin-conjugating enzyme E2N, (19) Fibroblast growth factor 21, (20) Adapter-related protein complex 2, (21) Ubiquitin-conjugating enzyme E2 variant 2, (22) IGFBP5, (23) Ubiquitin specific peptidase 15, (24) CD28 antigen, (25) Epsin 2, (26) Toll interacting protein, (27) von Hippel-Lindau syndrome homolog, (28) Ubiquitin specific peptidase 12, (29) SUMO / sentrin specific peptidase 2, (30) Heme oxygenase (decycling) 1, (31) Dual specificity phosphatases 18, (32) Ubiquitin specific peptidase 22, (33) Ubiquitin protein ligase E3A, ( 34) Protein phosphatase 1, (35) Hect domain and RCC1-like domain, (36) Calpastatin, (37) RAB GTPase activating protein 1, (38) Ubiquitin-conjugating enzyme E2L3, (39) interleukin 13 receptor alpha1, (40 ) RAD23a homolog (S. cerevisiae), (41) Ubiquitin specific peptidase 9, (42) Ubiquitin fusion degradation 1 like, (43) protein phosphatases 4 catalytic subunit, (44) ubiquitin-conjugat ing enzyme E2D3, (45) IGFBP6, (46) Sphingosine phosphate lyase 1, (47) adapter protein complex AP-2 alpha2 subunit, (48) RAB10, (49) HECT domain containing 1, (50) Ubiquitin specific peptidase 14, (51) Ubiquitin specific peptidase 32, (52) interleukin-1 receptor associated kinase 2, (53) ubiquitin-conjugating enzyme E2M, (52) adapter-related protein complex 3, (53) Ubiquitin specific peptidase 4, and (54) Dual specificity phosphatases 4.
[Aspect 5] The method according to any one of Aspects 1 to 4, wherein the exercise stimulus is caused by a pseudo exercise stimulus loaded on a muscle.
[Aspect 6] The method according to Aspect 4, wherein the pseudo motor stimulus is an electric pulse stimulus.
[Aspect 7] The method according to any one of Aspects 1 to 6, wherein the muscle is a cultured myotube cell.
[Aspect 8] The method according to any one of Aspects 1 to 7, wherein the expression level of the gene is measured at a stage after transcription of the gene.
[Aspect 9] The method according to Aspect 8, wherein the expression level of the gene is measured by Northern blotting, RT-PCR, real-time PCR, in situ hybridization, or microarray method.
[Aspect 10] The method according to any one of Aspects 1 to 7, wherein the expression level of the gene is measured at a stage after translation of the gene.
[Aspect 11] The method according to Aspect 10, wherein the gene expression level is measured by Western blotting, RIA, ELISA, or immunostaining.
[Aspect 12] A gene expression measurement kit for use in the method according to any one of Aspects 1-11.
[Aspect 13] Aspect including a probe or primer targeting a nucleotide comprising a nucleotide sequence of at least one gene selected from the gene group consisting of 54 types of genes according to Aspect 1 or a nucleotide comprising a partial sequence thereof 10. The gene expression measurement kit according to 10.
[Aspect 14] Insulin-dependent sugar uptake or GLUT4 membrane in muscle by exercise stimulation, containing as an active ingredient an expression product of at least one gene selected from the gene group consisting of 54 types of genes according to Aspect 1 A pharmaceutical composition having an action of enhancing or suppressing the amount of migration.
[Embodiment 15] Insulin-dependent sugar in muscle by muscle stimulation, containing as an active ingredient an expression product of at least one gene selected from the gene group consisting of CXC ligand 1, CXC ligand 4, and CXC ligand 5 A pharmaceutical composition having an action of enhancing uptake or GLUT4 membrane migration.
[Aspect 16] A pharmaceutical composition containing an expression product of Wnt inhibitory factor-1 (WIF-1) as an active ingredient and having an action of inhibiting insulin-dependent sugar uptake or GLUT4 membrane translocation in muscle by exercise stimulation .
[Aspect 17] The pharmaceutical composition according to any one of Aspects 14 to 16, which is a therapeutic agent or a prophylactic agent that simulates or enhances an exercise-dependent metabolic improvement effect.
[Aspect 18] The method comprises measuring the expression level of a gene by the method according to any one of Aspects 1 to 11 in the presence or absence of a test substance, and selecting a substance that varies the expression level. A method of screening for a substance that modifies the action of enhancing or suppressing the insulin-dependent sugar uptake into muscle or the amount of GLUT4 membrane transfer by muscle stimulation.
[Aspect 19] Screening for a substance that modifies insulin-dependent sugar uptake into muscle or enhancement of GLUT4 membrane translocation amount by exercise stimulation, wherein the gene is CXC ligand 1, CXC ligand 4 and / or CXC ligand 5. The method according to embodiment 18.
[Aspect 20] Aspect 18, wherein the gene is Wnt inhibitory factor-1 (WIF-1), and a substance that modifies insulin-dependent sugar uptake into muscle or suppression of the amount of GLUT4 membrane transfer by muscle stimulation is screened the method of.

As shown in the Examples, a group of genes whose expression level fluctuates (enhanced / decreased) in response to electrical pulse stimulation (pseudo-motor stimulation) in a special cultured muscle cell system developed by the present inventors was found. . Among these genes, in particular, a detailed analysis focusing on the secreted protein group revealed that the amount of GLUT4 membrane translocation, which is a key factor for insulin and exercise-dependent sugar metabolism, depends on the secreted protein. I found out that it was modified.

As a result, it is possible to measure exercise intensity and exercise by quantitatively or qualitatively measuring fluctuations in the expression level of the gene group and the protein group produced by the gene group (amount increased or attenuated, or their degree). Effects on muscle metabolism can be detected in a minimally invasive manner.

Furthermore, by using them as active ingredients, it is possible to simulate the effect of improving muscle metabolism of exercise or to further enhance the effect of improving muscle metabolism of exercise.

  The gene itself whose expression level is measured by the method of the present invention is publicly known. However, when a motor stimulus represented by an electric pulse stimulus (pseudo motor stimulus) or the like is applied, or during non-exercise or low exercise Compared with muscle / blood during strong exercise, the expression level is increased / decreased for the first time by the present inventors. These genes not only contribute to the formation and maintenance of healthy muscles, but also to measure exercise intensity that improves metabolism such as sugars and lipids, as shown in the examples below. Is a group of genes that can be key molecules.

Each of the above gene groups is a gene group that encodes secreted proteins secreted extracellularly and intracellular proteins (enzymes). Secreted proteins and intracellular proteins (enzymes) used in the present invention include mammalian, vertebrate, eukaryotic and prokaryotic secreted protein homologues including humans, rats and mice.

In the method of the present invention, in order to detect the intensity of the exercise stimulus loaded on the muscle, the expression status of each gene in various tissues, body fluids, organs, etc. including blood existing in the muscle or the vicinity thereof is determined. It is necessary to investigate. Here, “muscle” is a concept that includes not only living muscles (in vivo muscles) but also cell lines such as primary cultured muscle cells, cultured muscle cells, or cultured myotube cells. The “muscle” is preferably derived from mammals including humans, but muscles of other animal species can also be used. Further, the movement stimulation may be provided by “pseudo movement stimulation” represented by an electric pulse stimulation or the like performed by any method known to those skilled in the art as shown in the embodiments. In particular, when the method of the present invention is carried out using a cell line, the change in gene expression level can be measured by applying such a pseudo-stimulation to the cell line.

In order to detect the intensity of such motor stimulation, at least one of the above gene group, preferably at least any two genes, for example, CXC ligand 1 and CXC ligand 5, more preferably at least any An appropriate number, for example, a combination of 5 types of genes, most preferably all types of genes may be measured.

In the screening method of the method of the present invention, the expression level of each gene can be measured at any stage such as transcription and translation by any method known to those skilled in the art.

  For example, in the measurement after the transcription, in the screening method of the present invention, the expression level of each gene can be measured by the amount of mRNA or cDNA encoding it. Such mRNA is measured by various PCR methods such as RT-PCR method and real-time PCR method using primers designed appropriately based on the base sequence of each gene (DNA), Northern blot method, in situ hybridization method, and It can be performed by a method known to those skilled in the art, such as a microarray method (DNA chip) method.

  Alternatively, the measurement at the post-translational stage can be performed by Western blotting, RIA, ELISA, immunostaining, or the like. As described above, any of the methods listed here can be performed by those skilled in the art by any known method.

  The gene expression measurement kit used in the method of the present invention can take an appropriate configuration according to the specific measurement principle of each gene expression level. The kit targets, for example, a reagent comprising an antibody that specifically recognizes the protein of the present invention, or a nucleotide containing the nucleotide of the above gene or a partial sequence thereof for the measurement of the above mRNA or cDNA. , Amplification primers and hybridization probes. These primers or probes have an appropriate length, for example, 10 to 100 consecutive base sequences depending on the application.

  Various primers, probes, or antibodies included as components in the above kit may be labeled with an appropriate labeling substance such as any radioactive substance, fluorescent substance, and dye known to those skilled in the art. Further, the kit includes other elements or components known to those skilled in the art, such as various reagents, enzymes, buffers, reaction plates (containers), and the like, depending on the configuration and purpose of use.

Here, each of the above genes has a gene name and an accession number as described in Table 2 to Table 5 below, and the nucleotide sequence information thereof can be easily obtained from the database of each depository institution. It can be obtained. Here, the genes shown in Table 2 are increased (increased) in response to exercise stimuli, whereas the genes shown in Tables 3 to 5 are attenuated (decreased) in response to exercise stimuli. ) From the left, the columns in each table indicate the gene name, the accession number, and how many times (> 1) it was boosted (<1) or attenuated by electrical pulse stimulation.

  Furthermore, the DNA (genomic DNA and cDNA) encoding each of the genes in the present invention is complementary to the DNA (or cDNA corresponding thereto) in addition to the DNA consisting of the specific base sequence. DNA that hybridizes with DNA comprising a base sequence under stringent conditions, and that substantially encodes a protein having at least one action of each gene is included.

  Here, the hybridization is performed by, for example, Molecular cloning third. ed. (Cold Spring Harbor Lab. Press, 2001), or Current protocols in molecular biology (described by Fredick M. Ausubel et al., 198, et al., Et al., 198). In the case of using a commercially available library, it can be carried out according to the method described in the attached instruction manual.

  In this specification, “stringent conditions” means, for example, at a temperature of 60 ° C. to 68 ° C., a sodium concentration of 15 to 900 mM, preferably 15 to 600 mM, more preferably 15 to 150 mM, and pH of 6 to 8. Can be mentioned.

  Therefore, as a DNA that can hybridize under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence represented by the above sequence number, for example, homology with the entire base sequence of the DNA Examples thereof include DNA containing a base sequence having an average of about 80% or more, preferably about 95% or more, and more preferably 99% or more as a whole on average.

  Such DNA can be obtained, for example, by PCR or the like using appropriate primers prepared based on the base sequence information of the above databases. The DNA of the present invention can also be chemically synthesized by any method known to those skilled in the art.

  In addition to the polypeptide (protein) encoded by the nucleotide sequence of each of the above databases, the gene of the present invention has one or several amino acids deleted, substituted, or added in such an amino acid sequence. Also included is a protein consisting of an amino acid sequence (amino acid sequence having sequence homology with the above amino acid sequence) and having substantially at least one action of each gene.

  In order to determine sequence homology between two base sequences or amino acid sequences, the sequences are pre-processed to the optimal state for comparison. For example, by making a gap in one sequence, the alignment with the other sequence is optimized. Thereafter, the amino acid residues or bases at each site are compared. When the same amino acid residue or base as the corresponding site in the second sequence is present at a site in the first sequence, the sequences are identical at that site. Sequence homology between the two sequences is expressed as a percentage of the total number of sites (all amino acids or all bases) of the number of sites that are identical between the sequences.

  In accordance with the above principles, sequence homology between two base or amino acid sequences can be determined, for example, by the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990 and Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993). A BLAST program or FASTA program using such an algorithm is mainly used for searching a database for sequences showing high sequence homology to a given sequence. These are available, for example, on the website of the US National Center for Biotechnology Information.

  A DNA showing the sequence homology of the base sequence as described above or the sequence homology of the encoded amino acid sequence can be easily designed and prepared by a person skilled in the art based on the base sequence information of each database. is there.

Further, as shown in the examples of the present specification, the expression products of the above genes, that is, various proteins encoded by the genes, particularly secreted proteins, are the insulin-dependent sugars in muscles subjected to exercise stimulation. It has the effect of enhancing or suppressing uptake or GLUT4 membrane migration. Therefore, at least one gene product selected from the above gene group is useful as an active ingredient in a pharmaceutical composition that is a therapeutic or prophylactic agent that mimics or enhances an exercise-dependent metabolic improvement effect. Such a pharmaceutical composition can contain, for example, at least one of the above genes selected from C-X-C ligand 1, C-X-C ligand 4, and C-X-C ligand 5, preferably at least any three types.

  Such “proteins” include various recombinant proteins and peptides known to those skilled in the art that are prepared for the purpose of mimicking the physiological action of secreted proteins. These proteins are easily prepared by any method known to those skilled in the art using, for example, an appropriate recombination vector in which a gene (nucleic acid molecule) encoding the protein is expressed. be able to.

  Here, the “recombinant vector” includes a plasmid or a virus. In general, a recombinant vector contains an origin of replication, an expression control sequence such as a promoter, and a special gene that allows selection when the recombinant vector is introduced into a cell. Suitable vectors for the present invention include pMSXND expression vectors, retroviral vectors, adenoviral vectors and the like for mammalian cell systems. Moreover, those skilled in the art can very easily select appropriate vectors other than these for carrying out the present invention.

  Further, “expressible” means that a nucleic acid molecule (DNA) encoding a predetermined amino acid sequence has the ability to express a protein having the amino acid sequence under a predetermined condition. When a DNA encoding a predetermined amino acid sequence is operably linked, the DNA expresses a predetermined protein under predetermined conditions. Specifically, the DNA is linked to expression control sequences in the vector. Here, “expression control sequence” means a nucleic acid sequence that regulates the expression of other nucleic acid sequences, and also controls and regulates the transcription and preferably translation of other nucleic acid sequences. Expression control sequences include appropriate promoters, enhancers, transcription terminators, initiation codons (ie, ATG) in genes encoding proteins, splicing signals for introns, polyadenylation sites, stop codons, and the like.

  “Promoter” means a minimal sequence necessary for transcription. Promoters also include promoter elements that control gene expression in a cell-type-specific, tissue-specific, or externally-dependent manner by means of signals and regulators. The promoter element is bound to either the 5 'region or the 3' region of the expressed DNA. Promoters include both constitutive (constitutive) and inducible promoters. In order to stably express the constitutively active mutant and to obtain the effect of the present invention more effectively, the promoter is constitutive. A typical promoter is preferred.

When a mammalian cell is used as a host cell for a transformant, a promoter derived from a mammalian cell (for example, a metallothionein promoter), a promoter derived from a mammalian virus (for example, a retroviral terminal repeat (LTR), Adenovirus late promoter, vaccinia virus 7.5K promoter, etc.) can be used.

The introduction of the above recombinant vector into a host cell can be performed by any method known to those skilled in the art. For example, the desired DNA can be introduced by the calcium phosphate method, microinjection, electroporation, liposome, viral vector, various transfection reagents, and the like.

  The pharmaceutical composition of the present invention can be formulated by a known pharmaceutical method. For example, a pharmacologically acceptable carrier or medium, specifically sterilized water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, sustained release agent, etc. It is conceivable to administer. The pharmaceutical composition of the present invention may be in the form of an aqueous solution, tablet, capsule, troche, buccal tablet, elixir, suspension, syrup, nasal solution or inhalation solution. The content of the compound of the present invention can be appropriately determined by those skilled in the art according to the therapeutic purpose, administration route, treatment target, and the like.

  Administration to a patient can be local or systemic by any route, depending on the nature of the active ingredient. For example, it can be performed percutaneously, intramuscularly, intraperitoneally, intravenously, intrathecally, or orally. For example, it is desirable to introduce it into muscle or its nearby tissue by any appropriate means. A person skilled in the art can appropriately select an appropriate dose according to the age, weight, symptom, administration method and the like of the patient. The dosage and administration method can be appropriately selected by those skilled in the art according to the tissue transferability of the active ingredient of the pharmaceutical composition of the present invention, the purpose of treatment, the patient's weight, age, symptoms, and the like. For example, it can be administered at least 1 nM or more, preferably 10 nM or more, more preferably 100 nM or more, more preferably 1 μM or more.

In addition, for the purpose of simulating or enhancing the exercise-dependent metabolic improvement effect, any known method for those skilled in the art can be used to express, introduce, introduce or introduce the protein encoded by the gene, its recombinant protein / peptide, or Suppression can be performed. Preferably, it is carried out using a secreted protein or an antibody to the secreted protein by normal intravascular or intramuscular injection. In addition, a method of directly introducing cells such as endogenous or exogenous macrophages that produce a large amount of secreted protein, a secreted protein muscle by a transformant using a gene expression system using a viral vector, etc. It is also possible to utilize an expression system in a nearby tissue, production in the body such as an antibody against the secreted protein, an inhibitor against the secreted protein, suppression of the secreted protein using RNA interference method, and the like.

  Furthermore, by measuring the expression level of each of the genes according to the method of the present invention in the presence or absence of the test substance, it is possible to screen for a substance that can vary the expression level of the gene. A substance screened by this method is a substance that modifies (changes) the action of enhancing or suppressing the insulin-dependent sugar uptake into muscle or the amount of GLUT4 membrane transfer by exercise stimulation. For example, substances that increase the expression of CXC ligand 1, CXC ligand 4, and / or CXC ligand 5 have the effect of promoting insulin-dependent sugar uptake into muscles by muscle stimulation or enhancement of GLUT4 membrane translocation. . On the other hand, substances that increase the expression of Wnt inhibitory factor-1 (WIF-1) have an action of promoting insulin-dependent sugar uptake into muscles or suppression of GLUT4 membrane transfer by exercise stimulation. Therefore, the substance selected by such screening is useful as a pharmaceutical agent as an active ingredient having the above action.

  Hereinafter, the present invention will be described in detail by way of examples. However, the technical scope of the present invention is not construed as being limited in any way by the description of the following examples, and those skilled in the art based on the description of the present specification. Any modification / modification method that can be easily conceived is within the scope of the present invention. Also, unless otherwise stated, the following examples are described in, for example, Sambrook and Maniatis, in Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel, FM et al., Current Protocols. This was carried out by a well-known method known to those skilled in the art described in Molecular Biology, John Wiley & Sons, New York, NY, 1995 and the like.

Myotubes were formed using a cultured myocyte system developed by the inventors as described above. In other words, mouse-derived myoblasts C2C12 (ATCC No. CRL1772) cultured until they became confluent in a culture solution in which 10% fetal bovine serum was added to Dulbecco's modified Eagle medium, Dulbecco's modified Eagle medium + 2% bovine serum + 200% amino acid enhancement Myotubes were formed by culturing in the culture medium (Table 1) for 6 days. The culture medium was exchanged every 24 hours. The myotubes were given a simulated motor stimulus (24h + EP) by applying an electrical pulse stimulus of 40V, 1Hz, 2ms, 24 hours. On the other hand, instead of culturing under electric pulse stimulation for 24 hours, myotube cells obtained by culturing for 4 hours in serum-free Dulbecco's modified Eagle medium without applying electric pulse stimulation were used as a control group (24h-EP). It was. Exercise stimulating effects include phosphorylation of AMP kinase and phosphorylation of AMP kinase substrate acetylcoenzyme A carboxylase, phosphorylation of Erk5, JNK, and p38, which are indicators of stretch stimulation, and increased sugar uptake, GLUT4 membrane translocation It was confirmed using the measurement of

As a result, phosphorylation of AMP kinase (FIG. 1A; phospho-AMPK) and acetyl CoA carboxylase (FIG. 1A; phospho-ACC), which is an AMP kinase substrate, were enhanced by electrical pulse stimulation. In addition, an increase in phosphorylation of Erk5, Erk1 / 2, and JNK in response to stretch stimulation was also observed (FIG. 1B). Moreover, as a result of measuring the amount of sugar taken up by the cells, it was found that the amount increased about 1.5 times by electrical pulse stimulation (FIG. 1C). Furthermore, as a result of measuring the amount of GLUT4 membrane transfer, it became clear that electrical pulse stimulation increases the amount of GLUT4 membrane transfer in the basal state and enhances insulin-dependent GLUT4 membrane transfer (Figs. 1D and E). . The above shows that a motion-like effect is induced in the electrical pulse stimulation, and it was confirmed that the electrical pulse stimulation is a “pseudo-motion stimulation”.

  Phospho-ACC antibody (# 07-303, Ser79, Upstate Biotechnology, Lake Placid) was used for phosphorylation of AMP kinase and phosphorylation of acetyl coenzyme A carboxylase, which is a substrate of AMP kinase, using the method described in Non-Patent Document 3. , NY) and Western blot analysis using Phospho-AMPK antibody (# 07-626, Thr172, Upstate Biotechnology, Lake Placid, NY).

In addition, phosphorylation of Erk5, Erk1 / 2, JNK, and p38 was performed by anti-phospho BMK1 / Erk5 antibody (T218 / Y220, Upstate # 07-507, Cross react with phospho Erk1 / 2), Anti-JNK antibody (T183, respectively). / Y185, T221 / Y223, Upstate # 07-175), anti-phospho p38 antibody (T180 / Y182, Upstate # 09-272), and the method described in Non-Patent Document 3.

Furthermore, the sugar uptake amount was modified based on a conventional 2-deoxyglucose uptake method (Kanzaki M and Pessin JE, J Biol Chem, 2001, Vol. 276 (45), 42436-42444). The method described in Kaikai 2006-296282 was used.

Further, the amount of GLUT4 film transfer used the method described in JP-A-2006-340637 as described below.

[Method for measuring membrane translocation activity of recombinant GLUT4]
The membrane transfer activity of recombinant GLUT4 is measured using differentiated cultured myotube cells. Specifically, for example, after the cells are fixed, the amount of the labeling substance at the extracellular site of recombinant GLUT4 present on the cell membrane, that is, the amount of recombinant GLUT4 transferred to the cell membrane is measured. Consists of. In this case, the amount of recombinant GLUT4 present on the cell membrane at a certain point in time is measured. In this method, for example, the amount of the labeling substance can be measured by an immunostaining method using an antibody against the labeling substance by a method known to those skilled in the art. More specifically, a secondary antibody to which a fluorescent dye such as rhodamine is bound is reacted, and the amount of fluorescence is measured using a confocal microscope or the like.

It is known that GLUT4 transferred to the cell membrane by an exogenous stimulus or the like then moves back into the cell by endocytosis. Therefore, as another method for measuring the membrane translocation activity of recombinant GLUT4, recombinant GLUT4 present on the cell membrane, and recombinant GLUT4 cells that have been taken into the cell again after migrating onto the cell membrane The amount of externally labeled substance can also be measured. In this case, it becomes possible to measure the total amount of recombinant GLUT4 transferred onto the cell membrane during a certain period. According to this method, for example, a method known to those skilled in the art, differentiated myotube cells are cultured in a culture solution containing an appropriate amount of an antibody against the labeling substance, for example, 0.01 to 500 μg / ml, and then the cell extraction is performed. The amount of the antibody contained in the liquid can be measured using an antigen-antibody reaction. For example, when the labeling substance is a radioactive substance, the amount of the labeling substance can be directly measured using a liquid scintillation counter or SDS-PAGE, and can be measured using a cell immunostaining method, ELISA (Enzyme-Linked Immunosorbent Assay). ), Immunoprecipitation, and Western blotting, etc., can be measured by any method known to those skilled in the art.

Next, the culture fluid of myotube cells (24h + EP) and the control group (24h-EP) that were given 24 hours of electric pulse stimulation as described above was collected, and the amount of secreted protein contained therein was determined. It was measured. In addition, the amount of each secreted protein is Mouse KC Quantikine ELISA kit (R & D systems, MKC00B), Mouse LIX DuoSet (R & D Biosystems, DY443), Mouse IL-6 ELISA Ready-SET-Go! (EBioscience, # 88-7064) ) Using the ELISA method described in the attached protocol.

In this way, C2C12 myotube cells to which pseudo-motion stimulation was given and C2C12 myotube cells to which no pseudostimulation was given (control group) were added with 0.2% collagenase to be detached from the bottom of the cell culture dish. Only multinucleated myotube cells that could not pass were collected by passing. Total RNA was extracted from the crudely purified C2C12 myotube cells using Trizol Reagent (Invitrogen, San Diego, CA, USA), and the expression of the gene encoding the secreted protein contained therein was analyzed using a DNA microarray known to those skilled in the art. . As a result of the analysis, it is clear that the expression of the genes shown in Table 2 is enhanced (increased) in response to the exercise stimulus, while the expression of the genes shown in Tables 3 to 5 is attenuated (decreased). It became. From the left, the columns in each table indicate the gene name, the accession number, and how many times (> 1) it was boosted (<1) or attenuated by electrical pulse stimulation.

Next, as a result of comparing the two groups with respect to the expression level of the protein encoded by the gene shown in Table 1, interleukin 6 (IL-6; Fig. 2A) and CXC ligand were obtained by electrical pulse stimulation (pseudo-motor stimulation). 1 (CXCL1 / KC; Fig. 2B), CXC ligand 5 (CXCL5 / LIX; Fig. 2C) and other chemokines were found to be significantly increased in secretion. In addition, these factors are not contained at all in the culture medium in which the cells are not cultured (DMEM + 2% CS). Furthermore, as a result of detailed analysis taking advantage of in vitro, production of CXCL1 and CXCL5 started at an extremely early time of 1 hour after the application of an electric pulse, while IL-6 expression level dependent on electric pulse stimulation. It was found that a long electric pulse stimulation of 12 hours or more is necessary for the increase (FIGS. 3A-C).

It was also revealed that the production of these secreted proteins is dependent on the electric pulse frequency (Hz). That is, at the frequency of 0.1 Hz, an increase in the amount of protein secretion dependent on electric pulse stimulation was hardly observed, but it was observed that the increase was clearly increased at 1 Hz. This indicates that the production amount of these protein groups is controlled in a precise response (positive correlation) to the electric pulse frequency, that is, the intensity of exercise (FIGS. 4A to 4C).

Furthermore, analysis of the details of the production of these secreted proteins revealed that these proteins are produced and secreted specifically only by exercise stimuli and not by other stimuli such as insulin stimuli or changes in extracellular sugar concentration. It became.

That is, differentiated C2C12 myotube cells were replaced with DMEM + 2% CS containing 5 mM glucose (LG) or 22.5 mM glucose (HG) under the condition where 100 nM insulin was added or not added, and 40 V, 2 ms, Electric pulse stimulation was performed at 1 Hz. The medium was collected after 6 hours or 24 hours, and the amount of secreted protein contained was measured. As a result, it was found that insulin stimulation and changes in extracellular sugar concentration did not change the secreted amounts of CXCL1 and CXCL5, and these secreted protein amounts were changed only by exercise (FIGS. 5A-D).

Furthermore, the production mechanism of these factors in response to exercise stimuli was investigated. First, for the purpose of analyzing the involvement of multiple MAP kinases, protein synthesis inhibitors (CHX; 10 μg / ml cycloheximide) and calcium chelators (EGTA; 2 mM EGTA) are used as electrical pulses (40 V, 1 Hz, 2 ms, 24 hours. The effect of the production of CXCL1 / KC or CXCL5 / LIX in response to the above was analyzed. As a result, it became clear that protein synthesis inhibitors (CHX) and calcium chelators (EGTA) suppress the production of these chemokines induced by electrical pulse stimulation (FIGS. 6A, B, D, E).

Next, the influence of various MAP kinase inhibitors on the production of CXCL1 / KC or CXCL5 / LIX in response to electric pulses was analyzed (FIGS. 6C and F). PD98059 (10 μM), which inhibits activation of ERK1 / 2, JNK inhibitor PHZ1264 (20 μM), and p38 inhibitor in the presence or absence of SB203580 (10 μM), 40 V, 1 Hz, 2 ms, Electric pulse stimulation was performed under conditions of 6 hours, and the amount of CXCL1 / KC or CXCL5 / LIX produced in the medium was measured. As a result, it was revealed that the MAP kinase inhibitor suppressed the production of these chemokines induced by electrical pulse stimulation (FIG. 6).

Moreover, when MAP kinase activation at this time was investigated, although activation in response to electrical pulse stimulation was observed for ERK and JNK, the activity of p38 was decreased by electrical pulse stimulation (FIG. 1B). Combined with the 30-minute treatment with EPS, the activation of p38 was enhanced compared to the control, and MAP kinase activated by EPS was also maintained or inactivated by a feedback system induced by EPS. The possibility of being strongly suggested. When EPS-dependent expression of phosphatase (dual specificity phosphatase) that dephosphorylates MAP kinase was examined, it was revealed that gene expression including DUSP4 and 18 was attenuated (Table 3). 5) Thus, it can be seen that electrical pulse stimulation, that is, pseudo motor stimulation, not only increases gene expression but also attenuates some gene expression. Therefore, activation of MAP kinase and phosphatase gene expression shown in Tables 3 to 5 that control the activation of MAP kinase can be an indicator of exercise effect.

Next, we confirmed the effect of the protein encoded by the gene whose expression was increased by pseudo-motor stimulation on glucose metabolism using insulin-dependent or independent glucose transporter 4 (GLUT4) membrane translocation as an index. . That is, 10 ng / ml CXCL1 / KC, 50 ng / ml CXCL5 / LIX, 25 ng / ml were added to C2C12 myotube cells expressing differentiated Myc tag-labeled GLUT4 (produced by the method described in JP-A- 2006-340637 ). 1 hour or 4 hours with CXCL4 / PF4, 10 ng / ml IL-6, 10 ng / ml WIF-1 (all purchased from R & D Biosciences), or 10 mM acetate (purchased from Wako Pure Chemical Industries, Ltd.), a ligand for GPR43 After stimulation for a period of time, stimulation was performed with 100 nM or 0 nM insulin for 60 minutes. The amount of GLUT4 membrane migration was measured by the method described in JP-A- 2006-340637 .

When CXCL1 / KC was added, the amount of GLUT4 membrane transfer in the ground state was not changed, but the insulin-dependent GLUT4 membrane transfer amount was enhanced. In addition, CXCL5 / LIX, CXCL4 / PF4, and IL-6 increased the amount of GLUT4 membrane migration in the ground state and promoted insulin-dependent GLUT4 membrane migration (FIGS. 7A-C). In particular, analysis of the enhancement of insulin-dependent GLUT4 membrane translocation in response to CXCL1 and CXCL5 concentrations revealed that the addition of about 1 ng / ml CXCL1 or CXCL5 enhanced insulin-dependent GLUT4 membrane translocation ( FIG. 7B, C).

On the other hand, WIF-1 increased the amount of GLUT4 membrane translocation in the ground state, but inhibited insulin-dependent GLUT4 membrane translocation, that is, exhibited an insulin resistance state (FIG. 7A). Acetate (a ligand of GPR43) tended to promote GLUT4 membrane migration in the ground state only after 1 hour treatment, but this effect was not seen with 4 hour treatment, and insulin-dependent GLUT4 It showed an inhibitory effect on membrane translocation (FIG. 7A). Thus, although such secretory protein group and its receptor group increase by exercise, it turns out that the role in the metabolism of a cell has both a promoting action and an inhibitory action.

It is known that exercise stimulation promotes glucose uptake into muscle, but these results indicate that proteins such as CXCL1, CXCL5, WIF-1, and GPR43 mediate part of the promotion. Strongly suggested. Furthermore, exercise stimuli are generally considered to enhance insulin-dependent sugar uptake, and CXCL1 and CXCL5 have also been shown to mediate some of their potentiating effects. On the other hand, it became clear for the first time that exercise also produces factors that cause insulin resistance (WIF1, GPR43), and the combined effects of these secreted proteins and their receptors are related to insulin responsiveness after exercise. It can be seen that Therefore, these exercise-dependent secreted proteins that control GLUT4 membrane translocation to muscle are proteins that play a very important role in muscle glucose metabolism, such as regulation of muscle glucose uptake and muscle glycogen content. I understand.

By using the present invention, it is possible to evaluate exercise intensity, metabolic changes, etc. very easily and with high reproducibility in muscles of animals including humans, muscles collected from these, and muscle cells established from these muscles. It becomes possible to do.

First, it becomes possible to simply and minimally evaluate exercises in muscles of animals including humans. By analyzing the expression status of these genes, it is possible to predict how much the exercise of specific intensity improves the individual's ability to metabolize muscles during normal or pathological conditions. As a result, even if the same amount of exercise is added, it is easy to evaluate exercise effects that have very large individual differences in the effects, and even during pathological conditions, it is possible to predict and provide information on specific individual exercise therapy It becomes possible. Furthermore, it becomes possible to evaluate the future morbidity risk to diseases such as diabetes.

In addition, by using the method of the present invention, evaluation of the effect on exercise and risk assessment of diabetes, obesity, etc., and improving the metabolic effect of the tissue and the whole body by imitating or enhancing the exercise effect It becomes possible.

In addition, administration of these secreted proteins and / or intracellular proteins (enzymes) into the blood or the vicinity of muscles, or gene expression systems using gene therapy, etc., can be used to simulate exercise-dependent metabolic capacity improvement effects. Alternatively, it can be further enhanced.

In addition, the method of the present invention makes it possible to predict the effect of exercise on poultry animals bred in the meat industry, and simply evaluate what kind of exercise enhances the value of poultry animals as a food ingredient. Or by using the enzyme protein or secreted protein of the present invention and their receptors, genes encoding them, and substances that modify their physiological actions, it is possible to increase the value as a foodstuff. Become.

Furthermore, when searching for the mechanism of exercise effects using cultured muscle cell systems, the use of secreted proteins according to the present invention, their receptors, their antibodies, genes encoding them, and substances that modify their physiological actions. It is also possible that part or all of the mechanism of exercise effects will be clarified, and part or all of the mechanism may contribute to the development of foods or drugs that mimic exercise-dependent metabolic changes.

Expression of exercise-like effect by electrical pulse stimulation. The result of measuring the change in intracellular signal and the amount of sugar uptake after C2C12 myotube cells were stimulated with electric pulse under conditions of 40V, 2ms, 1Hz, 24 hours. The analysis result of the secreted protein by electrical pulse stimulation is shown. The result of the time-dependent change of the amount of secretory protein which increases in an electrical pulse stimulus-dependent manner is shown. The change in the amount of secreted protein in response to the frequency of electrical pulse stimulation is shown. Immediately after the medium change of differentiated C2C12 myotube cells, electrical pulse stimulation was performed under the conditions of 40 V, 2 ms, 24 hours, and the frequency (Hz) shown in the figure, the cell culture medium was collected, and the amount of each secreted protein produced Was measured. Changes in the amount of secreted protein in response to electrical pulse stimulation or insulin and extracellular sugar concentrations. The analysis result of the change of the amount of secreted protein by electrical pulse stimulation in the presence of various inhibitors is shown. The influence which the protein secreted by the identified electrical pulse stimulation has on the GLUT4 translocation of C2C12 myotube cells is shown.

Claims (7)

How to measure the variation of CXC ligand 1 and / or CXC ligand 5 gene expression level, based on the increase, for detecting the intensity of the exercise stimulation by electrical pulse stimulation, which is loaded into the muscle in muscle. The method of claim 1, wherein the muscle is a cultured myotube cell. The method according to claim 1, wherein the expression level of the gene is measured at a stage after transcription of the gene. The method according to claim 3, wherein the gene expression level is measured by Northern blotting, RT-PCR, real-time PCR, in situ hybridization, or microarray method. The method according to claim 1, wherein the expression level of the gene is measured at a post-translational stage of the gene. The method according to claim 5, wherein the gene expression level is measured by Western blotting, RIA, ELISA, or immunostaining. Use in a method according to any one of claims 1 to 6 , comprising a probe or primer targeting a nucleotide comprising the base sequence of CXC ligand 1 and / or CXC ligand 5 or a nucleotide comprising a partial sequence thereof. A gene expression measurement kit.