Therapeutic agent for diabetes

Abstract

To provide a therapeutic agent for diabetes with high efficacy.SOLUTION: The present invention provides a therapeutic agent for diabetes containing GREB1 (Growth Regulation By Estrogen In Breast Cancer 1) or a nucleic acid encoding GREB1, a β cell proliferation agent containing GREB1 or a nucleic acid encoding GREB1, and an insulin production promotion agent containing GREB1 or a nucleic acid encoding GREB1.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a therapeutic agent for diabetes. [Background technology]

[0002] Blood glucose level, which is the concentration of glucose in the blood, is always regulated within a certain range by the action of various hormones such as insulin, glucagon, and cortisol. Among these hormones, insulin is the only one that can lower blood glucose levels, and is produced in the beta cells of the pancreas. When the number of beta cells decreases, the body becomes deficient in insulin, the glucose in the blood deviates from the normal range, and diabetes develops. In recent years, the number of diabetes patients has been rapidly increasing around the world. Approximately 90% of diabetes patients have type 2 diabetes.

[0003] Many drugs for treating type 2 diabetes have been developed so far, including insulin injections that replenish insulin in the body, drugs that stimulate remaining β cells in the body to secrete insulin, drugs that improve insulin sensitivity in muscle and fat cells, drugs that cause the kidneys to excrete sugar into the urine, drugs that delay or suppress sugar absorption in the intestine, and drugs that suppress sugar production in the liver. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2004 / 072282 [Patent Document 2] International Publication No. 2011 / 007882 [Non-patent literature]

[0005] [Non-Patent Document 1] The Journal of Biological Chemistry 1999, 274 (30): 21095-21103 [Non-Patent Document 2] Molecular and Cellular Biology 2004, 24(7): 2831-28412831-2841 [Non-licensed document 3] The American Journal of Pathology 2005, 167 (2): 327-336

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Non-licensed literature 9

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Non-licensed Document 11

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Non-licensed Document 14

[0006] None of the conventional diabetes treatment drugs can completely control insulin secretion, restore the decreased number of β cells, or restore blood glucose levels to normal. Therefore, there is a demand for more effective diabetes treatment drugs. Therefore, one of the objects of the present invention is to provide a highly effective diabetes treatment drug. [Means for solving the problem]

[0007] In order to eliminate insulin deficiency, which is the main cause of diabetes, the present inventors have studied the transcriptional regulation mechanism of insulin gene in pancreatic β cells and identified inducible cAMP early repressor (ICER), a factor that suppresses insulin gene transcription (see Non-Patent Document 1). The present inventors have also generated transgenic mice (ICER-Tg mice) that overexpress ICER specifically in β cells, and demonstrated that ICER-Tg mice have exhausted β cells, reduced insulin production, and develop severe diabetes from an early stage (see Non-Patent Document 2). Based on these results, the present inventors have established ICER-Tg mice as a model of severe type 2 diabetes and diabetic nephropathy that maintains hyperglycemia (see Non-Patent Documents 3 and 4 and Patent Document 1).

[0008] Furthermore, the inventors administered 17β-estradiol (E2), a type of estrogen, to male ICER-Tg mice, bringing the ratio of the male hormone androgen to the female hormone 17β-estradiol in the male ICER-Tg mice closer to that in female ICER-Tg mice, and found that this induced rapid proliferation of β cells in the pancreatic islets and pancreatic ducts, resulting in significant recovery from the pathological condition (see Non-Patent Document 5 and Patent Document 2).

[0009] The present inventors also found that even after ICER-Tg mice developed diabetic nephropathy, administration of 17β-estradiol to the mice restored sclerotic kidney tissues (glomeruli and tubules) (see Non-Patent Document 6). Furthermore, the present inventors found that administration of 17β-estradiol enhanced the expression of molecules involved in glucose uptake in skeletal muscles, such as AKT and Glut4, and promoted glucose uptake (see Non-Patent Document 7).

[0010] The present inventors considered that an effective strategy for treating diabetes would be to regenerate and maintain a sufficient amount of endogenous β cells in the body. However, the reason why 17β-estradiol causes β-cell proliferation was unclear. After extensive research, the present inventors found that 17β-estradiol induces the expression of GREB1 (Growth Regulation By Estrogen In Breast Cancer 1) in pancreatic islets, which causes β-cell proliferation and reduces blood glucose levels, and that GREB1 is effective as a therapeutic agent for diabetes.

[0011] GREB1 is a protein encoded by the GREB1 gene. GREB1 is a protein that is expressed as a target gene of estrogen signaling in breast cancer cell lines and has been identified as a transcriptional coactivator that binds to estrogen receptors in the nucleus to promote transcription. GREB1 has been reported to promote cell proliferation in breast and prostate cancer cells that are sensitive to sex hormones such as estrogen and androgen. However, the relationship between GREB1 and blood glucose concentration was previously unknown.

[0012] According to an aspect of the present invention, there is provided a therapeutic agent for diabetes comprising GREB1 or a nucleic acid encoding GREB1.

[0013] According to an aspect of the present invention, there is provided a therapeutic agent for diabetes comprising GREB1 or a nucleic acid encoding GREB1 as an active ingredient.

[0014] According to an aspect of the present invention, there is provided a therapeutic agent for treating diabetes, the active ingredient of which consists essentially of GREB1 or a nucleic acid encoding GREB1.

[0015] According to an aspect of the present invention, there is provided a therapeutic agent for diabetes, the active ingredient of which comprises GREB1 or a nucleic acid encoding GREB1.

[0016] According to an aspect of the invention there is provided the use of GREB1, or a nucleic acid encoding GREB1, in the manufacture of a medicament for the treatment of diabetes.

[0017] According to an aspect of the present invention, there is provided the use of an active ingredient comprising GREB1 or a nucleic acid encoding GREB1 in the manufacture of a medicament for the treatment of diabetes.

[0018] According to an aspect of the invention there is provided the use of an active ingredient consisting essentially of GREB1 or a nucleic acid encoding GREB1 in the manufacture of a medicament for the treatment of diabetes.

[0019] According to an aspect of the present invention, there is provided the use of an active ingredient comprising GREB1 or a nucleic acid encoding GREB1 in the manufacture of a medicament for treating diabetes.

[0020] According to an aspect of the present invention, there is provided a method for treating diabetes, comprising administering to a diabetic patient a therapeutic agent for diabetes comprising GREB1 or a nucleic acid encoding GREB1.

[0021] According to an aspect of the present invention, there is provided a method for treating diabetes, comprising administering to a diabetic patient a therapeutic drug for diabetes that contains GREB1 or a nucleic acid encoding GREB1 as an active ingredient.

[0022] According to an aspect of the present invention, there is provided a method for treating diabetes, comprising administering to a diabetic patient a therapeutic agent for diabetes, the active ingredient of which consists essentially of GREB1 or a nucleic acid encoding GREB1.

[0023] According to an aspect of the present invention, there is provided a method for treating diabetes, comprising administering to a diabetic patient a therapeutic drug for diabetes, the active ingredient of which consists of GREB1 or a nucleic acid encoding GREB1.

[0024] According to an aspect of the present invention, there is provided a β-cell proliferation agent comprising GREB1 or a nucleic acid encoding GREB1.

[0025] According to an aspect of the present invention, there is provided a β-cell proliferation agent comprising GREB1 or a nucleic acid encoding GREB1 as an active ingredient.

[0026] According to an aspect of the present invention, there is provided a β-cell proliferation agent, the active ingredient of which consists essentially of GREB1 or a nucleic acid encoding GREB1.

[0027] According to an aspect of the present invention, there is provided a β cell proliferation agent, the active ingredient of which comprises GREB1 or a nucleic acid encoding GREB1.

[0028] According to an aspect of the present invention, there is provided use of GREB1 or a nucleic acid encoding GREB1 in the manufacture of a β-cell proliferation agent.

[0029] According to an aspect of the present invention, there is provided use of an active ingredient comprising GREB1 or a nucleic acid encoding GREB1 in the manufacture of a β-cell proliferation agent.

[0030] According to an aspect of the present invention, there is provided the use of an active ingredient consisting essentially of GREB1 or a nucleic acid encoding GREB1 in the manufacture of a β-cell proliferation agent.

[0031] According to an aspect of the present invention, there is provided use of an active ingredient consisting of GREB1 or a nucleic acid encoding GREB1 in the manufacture of a β-cell proliferation agent.

[0032] According to an aspect of the present invention, there is provided a method for expanding β cells in a subject, comprising administering to the subject a β cell expanding agent comprising GREB1 or a nucleic acid encoding GREB1.

[0033] According to an aspect of the present invention, there is provided a method for proliferating β cells in a subject, comprising administering to the subject a β cell proliferating agent comprising GREB1 or a nucleic acid encoding GREB1 as an active ingredient.

[0034] According to an aspect of the present invention, there is provided a method for expanding β cells in a subject, comprising administering to the subject a β cell expanding agent whose active ingredient consists essentially of GREB1 or a nucleic acid encoding GREB1.

[0035] According to an aspect of the present invention, there is provided a method for proliferating β cells in a subject, comprising administering to the subject a β cell proliferating agent whose active ingredient consists of GREB1 or a nucleic acid encoding GREB1.

[0036] According to an aspect of the present invention, there is provided an insulin production-promoting agent comprising GREB1 or a nucleic acid encoding GREB1.

[0037] According to an aspect of the present invention, there is provided an insulin production-promoting agent comprising GREB1 or a nucleic acid encoding GREB1 as an active ingredient.

[0038] According to an aspect of the present invention, there is provided an insulin production-promoting agent, the active ingredient of which consists essentially of GREB1 or a nucleic acid encoding GREB1.

[0039] According to an aspect of the present invention, there is provided an insulin production-promoting agent, the active ingredient of which comprises GREB1 or a nucleic acid encoding GREB1.

[0040] According to an aspect of the present invention, there is provided use of GREB1 or a nucleic acid encoding GREB1 in the manufacture of an insulin production-promoting agent.

[0041] According to an aspect of the present invention, there is provided use of an active ingredient comprising GREB1 or a nucleic acid encoding GREB1 in the manufacture of an insulin production-promoting agent.

[0042] According to an aspect of the present invention, there is provided use of an active ingredient consisting essentially of GREB1 or a nucleic acid encoding GREB1 in the manufacture of an insulin production-promoting agent.

[0043] According to an aspect of the present invention, there is provided use of an active ingredient consisting of GREB1 or a nucleic acid encoding GREB1 in the manufacture of an insulin production enhancer.

[0044] According to an aspect of the present invention, there is provided a method for promoting insulin production in a subject, comprising administering to the subject an insulin production-promoting agent comprising GREB1 or a nucleic acid encoding GREB1.

[0045] According to an aspect of the present invention, there is provided a method for promoting insulin production in a subject, comprising administering to the subject an insulin production-promoting agent comprising GREB1 or a nucleic acid encoding GREB1 as an active ingredient.

[0046] According to an aspect of the present invention, there is provided a method for promoting insulin production in a subject, comprising administering to the subject an insulin production-promoting agent whose active ingredient consists essentially of GREB1 or a nucleic acid encoding GREB1.

[0047] According to an aspect of the present invention, there is provided a method for promoting insulin production in a subject, comprising administering to the subject an insulin production-promoting agent whose active ingredient consists of GREB1 or a nucleic acid encoding GREB1. Effect of the Invention

[0048] According to the present invention, a highly effective therapeutic drug for diabetes can be provided. [Brief description of the drawings]

[0049] [Figure 1] 1 is a graph showing the time course of blood glucose concentration in mice, with the graph plot showing the mean value ± standard error. [Diagram 2] FIG. 2 is a schematic diagram showing the four groups of mice used in the microarray analysis. [Diagram 3] FIG. 3 is a heat map showing gene expression patterns in pancreatic islets from the four mouse groups obtained by microarray analysis. [Figure 4]Figure 4 is a schematic diagram showing the method of extracting genes obtained by microarray analysis. The relationship between genes whose expression was significantly increased in transgenic mice implanted with 17β-estradiol (E2), genes whose expression was decreased, and genes that have estrogen response element (ERE) sites is shown. A total of 67 genes were significantly changed in the Tg-E2 group, of which 52 were upregulated and 15 were downregulated. A total of 260 genes had estrogen response element (ERE) sites, and two genes were common to both groups. [Diagram 5] FIG. 5 is a table showing the statistics of two genes whose expression was increased in transgenic mice implanted with 17β-estradiol and which also contain estrogen response element (ERE) sites. [Figure 6] FIG. 6 is a schematic diagram showing allelic changes during the generation of mice in which Greb1 is knocked out specifically in β cells. [Figure 7] FIG. 7 is a photograph showing the results of genotype analysis by electrophoresis after amplification of genomic DNA by PCR. [Figure 8] 8 is a graph showing the relative expression levels of mRNA in pancreatic islets of mice with β-cell-specific Greb1 knockout. The graph plots show the mean ± standard error. [Figure 9] FIG. 9 is a schematic diagram showing the experimental design of Example 3. [Figure 10] 10 is a graph showing the time course of blood glucose concentration in mice with β-cell-specific Greb1 knockout and implanted with 17β-estradiol. The graph plots show the mean ± standard error. [Figure 11] FIG. 11 is a graph showing the time course of cumulative survival rates in mice with β-cell-specific Greb1 knockout and implanted with 17β-estradiol. [Figure 12] FIG. 12 is a fluorescent micrograph of a pancreatic islet immunostained with anti-insulin and anti-glucagon antibodies. [Figure 13]FIG. 13 is a graph showing the proportion of β cells in pancreatic islets. [Figure 14] FIG. 14 is a graph showing the area of ​​β cells in pancreatic islets. [Figure 15] 15 is a schematic diagram showing the experimental design of Example 4. The center photograph shows a 17β-estradiol pellet, and the right photograph shows a 17β-estradiol pellet implanted adjacent to a very small amount of isolated pancreatic islets transplanted under the renal capsule. [Figure 16] 16 is a graph showing the time course of blood glucose concentration in mice administered streptozotocin (STZ). The graph plots show the mean ± standard error. [Figure 17] FIG. 17 is a photograph of kidneys implanted with isolated pancreatic islets and 17β-estradiol. [Figure 18] FIG. 18 is a photograph of a cross section of a kidney implanted with isolated pancreatic islets and 17β-estradiol. [Figure 19] FIG. 19 shows fluorescent micrographs of kidneys implanted with isolated pancreatic islets and 17β-estradiol. [Figure 20] FIG. 20 is a graph showing the relative expression level of Greb1 mRNA in pancreatic islets transplanted into the kidney. [Figure 21] FIG. 21 is a graph showing blood glucose concentrations in male wild-type mice, male transgenic mice (ICER-Tg mice) that express inducible early inhibitor of cAMP (ICER Iγ) specifically in pancreatic β cells, and male ICER-Tg mice implanted with 17β-estradiol. [Figure 22] FIG. 22 is a graph showing the relative expression levels of mRNA of Greb1 and Greb1-associated factor genes in pancreatic islets of male wild-type mice, male ICER-Tg mice, and male ICER-Tg mice implanted with 17β-estradiol. [Diagram 23]FIG. 23 is a graph showing the relative expression levels of mRNA of genes of cell cycle-related factors in pancreatic islets of male wild-type mice, male ICER-Tg mice, and male ICER-Tg mice implanted with 17β-estradiol. [Figure 24] FIG. 24 is a graph showing the time course of blood glucose concentration in female ICER-Tg mice. [Diagram 25] FIG. 25 is a graph showing the relative expression level of insulin mRNA in pancreatic islets of female ICER-Tg mice. [Figure 26] FIG. 26 is a graph showing the relative expression levels of mRNA of Greb1 and Greb1-associated factor genes in pancreatic islets of female ICER-Tg mice. [Figure 27] FIG. 27 is a graph showing the relative expression levels of mRNA of genes of cell cycle-related factors in pancreatic islets of female ICER-Tg mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] The following describes embodiments of the present invention. However, it should not be understood that the following embodiments limit the present invention. From this disclosure, various alternative embodiments, examples, and operating techniques will become apparent to those skilled in the art. It should be understood that the present invention encompasses various embodiments not described herein.

[0051] A diabetes treatment drug according to an embodiment includes GREB1 (Growth Regulation By Estrogen In Breast Cancer 1) or a nucleic acid encoding GREB1. A β-cell proliferation agent according to an embodiment includes GREB1 or a nucleic acid encoding GREB1. An insulin production promoter according to an embodiment includes GREB1 or a nucleic acid encoding GREB1.

[0052] The diabetes treatment drug, β-cell proliferation agent, or insulin production promoter according to the embodiment is administered to a human or non-human animal subject. When GREB1 is delivered to the subject's pancreatic islets, it proliferates the β-cells in the pancreatic islets, thereby promoting insulin production, normalizing glucagon levels, and lowering the subject's blood glucose level. Therefore, GREB1 is effective in treating diabetes in the subject. Diabetes includes, but is not limited to, type 1 diabetes, type 2 diabetes, diabetes due to other specific mechanisms or diseases, and gestational diabetes.

[0053] When the nucleic acid encoding GREB1 is delivered to a subject's pancreatic islet, it is introduced into the cells of the pancreatic islet and produces GREB1 in the cells of the pancreatic islet. The nucleic acid encoding GREB1 may be DNA or RNA. The RNA may be mRNA. The nucleic acid encoding GREB1 may be carried by a vector. The vector may be a viral vector or a non-viral vector.

[0054] Examples of viral vectors include, but are not limited to, retroviral vectors, adenoviral vectors, lentiviral vectors, adeno-associated virus (AAV) vectors, and Sendai virus vectors. Examples of non-viral vectors include, but are not limited to, plasmid vectors. The nucleic acid encoding GREB1 may be contained in a liposome.

[0055] GREB1 or a nucleic acid encoding GREB1 may be bound or fused to a protein or peptide that binds to a receptor on a pancreatic cell. By binding or fusing to a protein or peptide that binds to a receptor on a pancreatic cell, GREB1 or a nucleic acid encoding GREB1 can be efficiently delivered to a pancreatic cell. The pancreatic cell is, for example, a beta cell.

[0056] The receptor of the pancreatic cell is, for example, a receptor of the glucagon superfamily. The receptor of the glucagon superfamily is, for example, a glucagon-like peptide-1 (GLP-1) receptor. The protein that binds to the receptor of the pancreatic cell is, for example, a protein of the glucagon superfamily. The protein of the glucagon superfamily is, for example, GLP-1. The peptide of the glucagon superfamily is, for example, a peptide that has agonist activity against the receptor of the glucagon superfamily.

[0057] For example, the nucleophilic reactive portion of GREB1 may be reacted with an electrophilic reactive portion of a protein or peptide that binds to a receptor on pancreatic cells to bind GREB1 to the protein or peptide that binds to the receptor on pancreatic cells, or the electrophilic reactive portion of GREB1 may be reacted with a nucleophilic reactive portion of a protein or peptide that binds to a receptor on pancreatic cells to bind GREB1 to the protein or peptide that binds to the receptor on pancreatic cells.

[0058] Examples of nucleophilic reactive groups include, but are not limited to, amino, thiol, and hydroxyl. Examples of electrophilic reactive groups include, but are not limited to, carboxyl, acyl chloride, anhydride, ester, succinimide ester, alkyl halide, sulfonate ester, maleimide, haloacetyl, and isocyanate.

[0059] Alternatively, GREB1 can be linked to an electrophilic reactive portion of a protein or peptide that binds to a receptor on pancreatic cells via a linker, thereby linking GREB1 to a protein or peptide that binds to a receptor on pancreatic cells.

[0060] The therapeutic agent for diabetes according to the embodiment includes an effective amount of GREB1 or a nucleic acid encoding GREB1. The effective amount is, for example, an amount capable of treating diabetes. Whether or not diabetes can be treated can be determined, for example, by whether or not the subject's blood glucose concentration changes to approach a normal value. The effective amount may vary depending on the subject's weight, age, symptoms, pathology, and administration method.

[0061] The β cell proliferation agent and insulin production promoter according to the embodiments each contain an effective amount of GREB1 or a nucleic acid encoding GREB1. The effective amount is, for example, an amount capable of proliferating β cells or promoting insulin production.

[0062] The diabetes treatment drug, beta cell proliferation agent, or insulin production promoter according to the embodiment may constitute a pharmaceutical composition together with at least one selected from a pharma- ceutically acceptable excipient, carrier, buffer, and stabilizer.

[0063] The carrier may be a liquid. The liquid carrier may contain at least one selected from, for example, water, petroleum, animal oil, vegetable oil, mineral oil, and synthetic oil. The diabetes treatment drug, β-cell proliferation agent, or insulin production promoter according to the embodiment may be contained in an injection solution. Examples of the injection solution include, but are not limited to, sodium chloride injection solution, Ringer's injection solution, and lactated Ringer's injection solution.

[0064] The method of administration of the therapeutic agent for diabetes, the β cell proliferation agent, or the insulin production promoter according to the embodiment may be oral administration or parenteral administration. The therapeutic agent for diabetes, the β cell proliferation agent, or the insulin production promoter according to the embodiment may be locally administered to the pancreas, preferably to the pancreatic islets, of a subject.

[0065] Example 1 We prepared wild-type male mice (male WT mice) on the C57BL / 6 background, male transgenic mice (male ICER-Tg mice) in which inducible early inhibitor of cAMP (ICER Iγ) is highly expressed specifically in pancreatic β cells, and female transgenic mice (female ICER-Tg mice) in which ICER Iγ is highly expressed specifically in pancreatic β cells. ICER Iγ strongly suppresses the expression of insulin and cyclin A in β cells, reducing β cell proliferation. Therefore, ICER-Tg mice develop severe diabetes early after birth.

[0066] Mice were housed in an animal facility under a 12-h light / dark cycle. Mice were provided with water and standard diet (CE-2, CLEA Japan) ad libitum. The diet contained 8.9% water, 24.9% crude protein, 4.6% crude lipid, 4.1% crude fiber, 6.6% crude ash, and 51% soluble nitrogen-free matter, and contained 344.9 kcal per 100 g. The diet was suitable for maintaining normal blood glucose concentrations over the long term.

[0067] In this and the following examples, the body weight and post-breakfast glucose concentration of the mice were measured every two weeks. The glucose concentration in blood collected from the tail of the mice was measured using a blood glucose meter (ONE-TOUCH UltraVue, Johnson & Johnson or GLUCOCARD DIAmeter, registered trademark, ARKRAY). The glycosylated hemoglobin concentration (HbA1c) in blood collected from the tail of the mice was measured using a hematology analyzer (DCA2000, Siemens Healthineers).

[0068] As shown in Figure 1, the blood glucose concentration of male WT mice (n = 6) remained constant at a low level from 3 to 24 weeks after birth. Male ICER-Tg mice (n = 26) were severely diabetic, and blood glucose concentrations increased after birth and remained high thereafter. In contrast to male ICER-Tg mice, the blood glucose concentration of female ICER-Tg mice (n = 7) increased after birth but then decreased, becoming equivalent to that of male WT mice.

[0069] To induce proliferation of β-cells in pancreatic islets, 19-week-old male ICER-Tg mice (littermates, n = 6) were implanted with 0.72 mg of 17β-estradiol (E2) 90-day sustained-release pellets (Innovative Research of America) in the nape of the neck. One week after implantation, blood glucose levels were halved and continued to decrease thereafter. After 6 weeks (6 months of age), blood glucose levels had decreased to the same level as those in male WT mice and female ICER-Tg mice.

[0070] Next, as shown diagrammatically in Figure 2, pancreatic islets were isolated from 6-month-old male WT mice (n = 6), male ICER-Tg mice (n = 15) that had not been implanted with 17β-estradiol, male ICER-Tg mice (n = 6) that had been implanted with 17β-estradiol for 6 weeks, and female ICER-Tg mice (n = 7). Total RNA was extracted from the isolated islets using a reagent (TRIzol, Invitrogen or RNeasy Kit, Qiagen).

[0071] Labeled cRNA was prepared by amplified from total RNA using a cRNA preparation kit (Low Input Quick Amp Labeling Kit, Agilent Technologies). The cRNA was hybridized with a gene expression microarray (SurePrint G3 Mouse Gene Expression Microarray 8x60K, Agilent Technologies) containing comprehensive mouse coding RNA, and the microarray was scanned with a scanner (Agilent Technologies) to calculate the relative hybridization intensity for each gene. Furthermore, based on the results of the microarray analysis, a heat map showing the gene expression pattern shown in Figure 3 was generated using statistical analysis software (R) to profile gene expression in the pancreatic islets. In the color key of the heat map in Figure 3, the plus side indicates upregulation and the minus side indicates downregulation. The heat map showed that the expression of many genes was upregulated in male ICER-Tg mice 6 weeks after implantation of 17β-estradiol.

[0072] In male ICER-Tg mice 6 weeks after implantation of 17β-estradiol, the expression of 67 genes was significantly changed. Of these, the expression of 52 genes was increased and the expression of 15 genes was decreased. In addition, there were 260 genes with estrogen response region (ERE) sites that respond to 17β-estradiol. As shown in Figure 4, Greb1 and Nudt4 were two genes that were common to the group of genes whose expression was increased in male ICER-Tg mice implanted with 17β-estradiol and the group of genes with ERE sites.

[0073] The signal intensity of Greb1 in male ICER-Tg mice implanted with 17β-estradiol obtained by microarray analysis was compared with that in male WT mice, male ICER-Tg mice without 17β-estradiol implants, and female ICER-Tg mice. As shown in Figure 5, the signal intensity of Greb1 in male ICER-Tg mice implanted with 17β-estradiol was higher in terms of Z score and intensity ratio. The expression of Greb1 in male ICER-Tg mice implanted with 17β-estradiol was 4.6-fold higher than that in male ICER-Tg mice without 17β-estradiol implants, and 6.7 to 10-fold higher than that in wild-type mice. This result indicates that the increase in the concentration of 17β-estradiol in the body induces the expression of Greb1, which leads to the proliferation of β cells and the reduction of blood glucose concentration.

[0074] Example 2 C57BL / 6N-Greb1 tm1a(KOMP)Wtsi / BayMmucd mouse (Greb1 tm1a / tm1a We obtained C57BL / 6J-Ins1 mice from the Knockout Mouse Project (KOMP) repository at the University of California, Davis. We also obtained C57BL / 6J-Ins1 mice, a bicistronic Cre driver mouse with P2A-nls Cre knocked in immediately before the stop codon of the Ins1 gene. em1(cre)Utr Mice were obtained from the RIKEN BioResource Research Center. tm1a / tm1a Mouse and Ins1 em1(cre)Utr The mice were crossed to produce mice with Greb1 knockout in β cells (Ins1-Gre + / - ;Greb1 tm1a / tm1a In the Greb1 knockout mice, the Tm1b allele is generated in the pancreatic β cells, as shown diagrammatically in Figure 6.

[0075] Wild-type mice, Greb1 tm1a / tm1aGenomic DNA from mice and Greb1 knockout mice was amplified by PCR and the genotypes were analyzed by electrophoresis. As shown in Figure 7, the wild-type allele of 2018 bp was detected in the wild-type mice, and the Greb1 knockout mice were detected in the wild-type mice. tm1a / tm1a The wild-type allele and the 4212 bp Tm1a allele were detected in mice, and the wild-type allele and the 625 bp Tm1b allele were detected in Greb1 knockout mice.

[0076] 17-week-old female Greb1 tm1a / tm1a Pancreatic islets were isolated from 17-week-old female Greb1 knockout mice (n=10) and 17-week-old female Greb1 knockout mice (n=5), and total RNA was extracted from the islets. tm1a / tm1a The relative expression level of mRNA in Greb1 knockout mice was measured, with the expression level of mRNA in mice set at 1. As a result, as shown in Figure 8, it was confirmed that Greb1 was not expressed in the pancreatic islets of Greb1 knockout mice.

[0077] Example 3 As shown diagrammatically in FIG. 9, 114-week-old Greb1 tm1a / tm1a Control mice and 114-week-old Greb1 knockout (KO) mice were intraperitoneally administered 150 mg / kg streptozotocin (STZ, Sigma-Aldrich) to induce hyperglycemia by severely destroying β cells. One week after streptozotocin administration, Greb1 tm1a / tm1a 17β-estradiol (E2) was implanted into male Greb1 mice and Greb1 knockout mice, respectively. As shown in Figs. 10 and 11, male Greb1 mice tm1a / tm1a After implantation of 17β-estradiol, the blood glucose concentration of the mice (n=7) gradually decreased, and most of them did not die. In contrast, the blood glucose concentration of Greb1 knockout mice (n=7, including 3 females and 4 males) did not decrease, even after implantation of 17β-estradiol, and they gradually became weak, and most of them died. tm1a / tm1aThe difference in survival rate between the Greb1 knockout mice (n=19) and the Greb1 mice (n=14) was statistically significant.

[0078] Greb1 was injected 4 to 5 weeks after streptozotocin administration (19 weeks of age). tm1a / tm1a Greb1 was isolated from Greb1 knockout mice and Greb1 mice at 10 weeks of age (24 weeks of age). tm1a Pancreatic islets were isolated from mice and immunostained with anti-insulin and anti-glucagon antibodies as shown in Figure 12. As shown in Figure 13, Greb1 tm1a / tm1a In mice (analysis at 4 and 5 weeks: n = 3, analysis at 10 weeks: n = 4), the ratio of β cells to the total of α cells and β cells in the pancreatic islets increased over time. tm1a / tm1a In mice, the area of ​​β cells in the pancreatic islets increased significantly over time, and the morphology of many islets normalized. In contrast, in Greb1 knockout mice (n=7, including 4 females and 3 males), the proportion of β cells was low and the morphology of the pancreatic islets was disrupted, with no improvement observed. These results indicate that in the absence of Greb1, β cells cannot proliferate even when 17β-estradiol is implanted, indicating that Greb1 is a β cell growth factor.

[0079] Example 4 Eight-week-old wild-type (WT) mice were prepared, and two days later, 180 mg / kg streptozotocin (STZ, Sigma-Aldrich) was administered to the tail vein of the mice to significantly destroy β-cells and induce severe diabetes. Five days after preparing the mice, 50 isolated islets were transplanted into a single site under the kidney capsule of the streptozotocin-administered wild-type mice, as shown in Figure 15. It has been previously reported that transplantation of 50 isolated islets alone cannot improve hyperglycemia in diabetic mice. However, when 17β-estradiol (E2) pellets (1.5 mm diameter, 0.72 mg, 90-day sustained release, Innovative Research of America) were implanted near the 50 isolated islets under the kidney capsule, the mice did not die.

[0080] In two independent experiments, the blood glucose concentration of wild-type mice (n=7 for the first experiment, n=3 for the second experiment) rose sharply after administration of streptozotocin, as shown in Figure 16. After implantation of isolated islets and 17β-estradiol subcutaneously in the kidney, the blood glucose concentration decreased over 30 days, although with some fluctuation, and was then kept stable within the normal range. On the 46th day after the mice were prepared, the kidneys (one kidney) in which the islets were transplanted were removed from the mice (n=2), and the blood glucose levels of the mice immediately rose. These results indicate that the transplanted islets maintained normal blood glucose levels by exposure to 17β-estradiol, i.e., the maintenance of normal blood glucose was completely dependent on the transplanted islets.

[0081] Kidneys were removed on day 109 after mouse preparation. As shown in Figure 17, many new blood vessels were observed extending and spreading into the islet grafts and pellets. Sections of kidneys removed on days 109 and 145 after mouse preparation were fixed with 4% paraformaldehyde. The fixed kidney sections were stained with hematoxylin and eosin (HE). As shown in Figure 18, the islets expanded under the renal capsule. When the islets transplanted under the renal capsule were immunostained with insulin and BrdU or insulin and glucagon antibodies, the beta cells of the islets were observed to proliferate, as shown in Figures 19-1 and 19-2.

[0082] On the 109th day after the mice were prepared, kidneys were removed from the mice (n=3), the renal capsule was peeled off, and the islet grafts were isolated. Gene expression in the islet grafts was examined. As a result, as shown in Figure 20, the relative expression level of Greb1 in the islet grafts of mice that were not implanted with 17β-estradiol was set to 1, and the relative expression level of Greb1 in the islet grafts of mice that were implanted with 17β-estradiol was significantly increased.

[0083] Example 5 We prepared 6-month-old male WT mice (n=6), 6-month-old male ICER-Tg mice (n=15), and 6-month-old male ICER-Tg mice implanted with 0.72 mg of 17β-estradiol (E2) at 19 weeks of age (male ICER-Tg-E2 mice, n=6). The blood glucose concentration of each mouse was measured and the results are shown in Figure 21.

[0084] Figure 22 shows the results of measuring the expression levels of mRNA of Greb1 and Greb1-related factor genes in pancreatic islets isolated from each male mouse. Nik-specific kinase (Nrk) is a factor that is inversely correlated with Greb1 activity. Estrogen receptor α (ERα) and hepatic receptor homolog 1 (LRH1) are known to be induced in β cells in rapid response to an increase in 17β-estradiol. In breast cancer cell lines, ERα and LRH1 are known to activate GREB1 expression.

[0085] The graph in Figure 22 shows the relative expression levels of mRNA in male ICER-Tg mice and male ICER-Tg-E2 mice, with the expression level in male WT mice set at 1. In male ICER-Tg-E2 mice, the expression of Greb1 was significantly increased. The expression of LRH-1 was also increased, although it was lower than that of Greb1. The expressions of ERα and Nrk were very low. LRH-1 was highly expressed not only in male ICER-Tg-E2 mice but also in male ICER-Tg mice.

[0086] Furthermore, the results of measuring the expression levels of mRNA of genes of factors related to the cell cycle in pancreatic islets isolated from each male mouse are shown in Figure 23. The graph in Figure 23 shows the relative expression levels of mRNA in male ICER-Tg mice and male ICER-Tg-E2 mice, with the expression level of mRNA in male WT mice set at 1. Compared with male WT mice and male ICER-Tg mice, the expression of genes encoding cyclin A2, cyclin B1, cyclin B2, and aurora kinase B was significantly increased in male ICER-Tg-E2 mice.

[0087] These results indicate that exogenous 17β-estradiol increased the expression of Greb1, LRH1, cyclin A2, cyclin B1, cyclin B2, and aurora kinase B. In addition, the increased expression of cell cycle-related factors suggests that β-cell proliferation occurred.

[0088] Example 6 Female ICER-Tg mice aged 5 weeks, 8 to 12 weeks, 13 to 18 weeks, and 24 weeks were prepared. Blood glucose concentrations of each female mouse were measured and are shown in FIG. 24. Although blood glucose concentrations of 5-week-old female ICER-Tg mice were high, blood glucose concentrations decreased with age and reached normal concentrations from 13 weeks of age onwards.

[0089] It has been reported that in female wild-type rats, the concentration of endogenous 17β-estradiol (E2) in plasma gradually increases from 3 to 4 weeks after birth, further increases at 6 weeks, and then gradually decreases by 6 months of age. Therefore, it was suggested that the decrease in blood glucose concentration in female ICER-Tg mice corresponds to the increase in the concentration of endogenous 17β-estradiol in plasma.

[0090] Pancreatic islets were isolated from each female ICER-Tg mouse, and the expression level of insulin mRNA was measured. The expression level of insulin mRNA in female ICER-Tg mice is shown in the graph of FIG. 25, with the expression level of insulin mRNA in male mice aged 13 to 15 weeks set as 1. In female ICER-Tg mice, the expression level of insulin mRNA significantly increased with age. The increase in the expression level of insulin mRNA reflects an increase in β cells.

[0091] We also measured the expression levels of mRNA for Greb1 and Greb1-related factor genes in pancreatic islets isolated from each female ICER-Tg mouse. The relative expression levels of mRNA in female ICER-Tg mice are shown in the graph in Figure 26, with the expression levels of mRNA in 13- to 15-week-old male mice set to 1.

[0092] In 5-week-old female ICER-Tg mice, the expression of ERα and LRH-1 was high, whereas the expression of Greb1 and Nrk was low. In 8- to 12-week-old female ICER-Tg mice, the expression of ERα was significantly decreased, whereas the expression of Greb1 increased by approximately 8-fold. In 13- to 18-week-old female ICER-Tg mice, where blood glucose levels had decreased sufficiently and insulin expression had increased, the expression of ERα and LRH-1 was further decreased, and the expression of Greb1 was also decreased, whereas the expression of Nrk was increased. In 24-week-old female ICER-Tg mice, the expression of ERα, LRH-1, Greb1, and Nrk was decreased. Thus, it was shown that the expression of Greb1 is transient.

[0093] In addition, we measured the expression levels of mRNA of genes of factors related to the cell cycle in pancreatic islets isolated from each female ICER-Tg mouse. The expression levels of mRNA in 13- to 15-week-old male mice were set as 1, and the relative expression levels of mRNA in female ICER-Tg mice are shown in the graph in Figure 27.

[0094] All cell cycle-related factors measured except for cyclin D1 and cyclin E2, cyclin A2, cyclin B1, cyclin B2, cyclin D2, cyclin E1, Aurora kinase A, and Aurora kinase B, increased at 8 to 12 weeks of age, when blood glucose concentrations decline, and then decreased at 13 to 18 weeks of age, when insulin levels increase sufficiently.

[0095] These results indicate that in female ICER-Tg mice, increased endogenous 17β-estradiol increased the expression of Greb1, cyclin A2, cyclin B1, cyclin B2, cyclin D2, cyclin E1, Aurora kinase A, and Aurora kinase B. These results also suggest that increased expression of cell cycle-related factors led to β-cell proliferation.

Claims

1. A therapeutic agent for diabetes, comprising GREB1 (Growth Regulation By Estrogen In Breast Cancer 1) or a nucleic acid encoding GREB1.

2. A β-cell proliferation agent comprising GREB1 (Growth Regulation By Estrogen In Breast Cancer 1) or a nucleic acid encoding GREB1.

3. An insulin production-promoting agent comprising GREB1 (Growth Regulation By Estrogen In Breast Cancer 1) or a nucleic acid encoding GREB1.

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