High expression of ffar4 in adipocytes and uses thereof

Abstract

The present invention provides a novel method for the treatment and / or prevention of various diseases, such as age-related impaired glucose tolerance, impaired cognitive ability. The present invention relates to a fat cell in which FFAR4 is highly expressed, a transplantation composition containing the same, and a method for treating and / or preventing various diseases using the transplantation composition, for the treatment and / or prevention of various diseases, particularly age-related impaired glucose tolerance and age-related impaired cognitive ability. Further, the present invention relates to a transgenic mouse in which FFAR4 is highly expressed.

Description

Technical Field

[0001] This invention relates to adipocytes that highly express FFAR4 and are useful in the treatment and / or prevention of various diseases, as well as transplantation compositions containing these cells and methods for treating and / or preventing various diseases in animals and humans using the transplantation compositions. Target diseases in this invention include, for example, age-related glucose intolerance and cognitive impairment. Background Technology

[0002] It is known that glucose tolerance impairs with age, making older adults more prone to postprandial hyperglycemia (blood glucose levels exceeding 140 mg / dL 2 hours after meals), thus increasing their risk of developing diabetes. The prevailing view is that preventing diabetes in the elderly lies in controlling postprandial blood glucose levels; in other words, it is believed that treatments to improve age-related impaired glucose tolerance are effective. However, concerns remain regarding the potential side effects of existing diabetes medications, such as hypoglycemia, and a safe treatment method has not yet been established.

[0003] G protein-coupled receptors (GPCRs) are receptors with a characteristic molecular structure that penetrates the cell membrane seven times. The target molecules for drugs treating most diseases, including hypertension, arrhythmia, angina pectoris, asthma, and peptic ulcers, belong to this gene family. Therefore, GPCRs have attracted considerable attention as target molecules for drug discovery. GPCRs are activated from outside the cell via specific ligands, transmitting their information to intracellular G proteins to produce physiological activity. It is believed that the characteristic molecular structure of GPCRs undergoes significant structural changes upon binding to specific ligands, leading to the activation of G proteins.

[0004] FFAR4 (formerly known as GPR120, with the same amino acid sequence as NP_859529 registered in GenBank) was found to be one of the GPCRs. It activates free fatty acid receptors for intracellular signal transduction using free fatty acids (especially ω-3 fatty acids such as DHA and EPA, which are essential fatty acids) as ligands. Its physiological function, promoting the secretion of glucagon-like peptide GLP-1 in the intestine under the stimulation of free fatty acids, has been clarified (Hirasawa, A. et al. Nat. Med. 11, 90-94, 2005 (Non-Patent Literature 1)). Furthermore, by combining fluorescent ligand and flow cytometry ligand-receptor interaction analysis methods, the interaction of FFAR4 with long-chain fatty acids has been confirmed (Sun, Q et al. Mol. Pharmacol. 78, 804-810, 2010 (Non-Patent Literature 2)). Furthermore, FFAR4 knockout mice exhibited phenotypes including adipocyte hypertrophy, increased adipose tissue weight, weight gain, fatty liver, and impaired glucose tolerance. This phenomenon is attributed to inhibited adipose tissue differentiation and reduced fatty acid synthesis (Ichimura, A. et al. Nature 483, 350-354, 2012 (Non-Patent Literature 3)). Thus, FFAR4, as a fatty acid sensor, has been shown to be closely related to dietary obesity. For dietary obesity, the application of FFAR4-targeted preventative and therapeutic drugs is anticipated (Hara, T. et al. Rev Physiol Biochem Pharmacol. 164, 77-116, 2013; Milligan, G. et al. Trends Pharmacol Sci. 38, 809-821, 2017 (Non-Patent Literature 4, 5)).

[0005] To date, the application of FFAR4-targeted preventive and therapeutic drugs has tended towards searching for compounds with FFAR4 agonist activity. Research is underway to find compounds with FFAR4 agonist activity that are useful for the treatment and / or prevention of diabetes, obesity, and hyperlipidemia (Japanese Patent Application Laid-Open No. 2012-520240 (Patent Document 1), Japanese Patent Application Laid-Open No. 2008-001690 (Patent Document 2), International Publication No. 2005 / 083070 (Patent Document 3)). Furthermore, although cells with the gene encoding FFAR4 have been created, ligand screening and activity analysis have not been conducted (Japanese Patent Application Laid-Open No. 2016-214135 (Patent Document 4), Japanese Patent Application Laid-Open No. 2012-520240 (Patent Document 1), Japanese Patent Application Laid-Open No. 2008-001690 (Patent Document 2)), and there has been no attempt to use cells with the gene encoding FFAR4 for therapeutic purposes. Furthermore, even for this analytical purpose, the FFAR4 analysis in adipocytes was performed using cells that had been induced to differentiate from mouse-derived cultured cells 3T3-L1 into adipocytes. The results showed that this method used FFAR4 expression inhibition caused by induced differentiation based on the addition of FFAR4 agonists, siRNA, etc., and there are currently no known examples of FFAR4 being highly expressed (overexpressed) in adipocytes.

[0006] International Patent Publication No. 2003 / 106663 (Patent Document 5) discloses an ex vivo gene therapy method using adipocytes with introduced foreign genes. However, in this invention, the transplanted adipocytes are used in vivo to produce proteins encoded by the introduced foreign genes (insulin, a hormone called GLP-1) like a manufacturing plant, and secrete them extracellularly. There is no consideration given to transplanting adipocytes that express receptors that can penetrate cell membranes.

[0007] International Patent Publication No. 2005 / 083070 (Patent Document 3) describes an invention related to a composition for lowering blood glucose levels containing a "GT01 polypeptide". However, the "GT01 polypeptide" in this invention refers to a G protein-coupled receptor distributed on the surface of enteroendocrine cells or enteroendocrine cell lines, which transmits signals for GLP-1 secretion into the cell through the binding of its ligands, and is not intended to be expressed on the surface of adipocytes. It should be noted that there are no reports of FFAR4 promoting GLP-1 secretion in adipocytes. In addition, although Patent Document 3 mentions ex vivo treatment as a gene therapy composition, it does not disclose specific gene delivery techniques, and of course, makes no mention of delivering genes to adipocytes, adipose stem cells, or preadipocytes.

[0008] In addition, the maintenance and improvement of cognitive function are required for all age groups, from young people to the elderly. In particular, the maintenance and improvement of memory, thinking ability, and judgment are very important in daily life. They are related to preventing the decline of cognitive function with aging, prolonging healthy lifespan, and inhibiting the decline of quality of life.

[0009] To date, various proposals have been put forward regarding compositions for maintaining or improving cognitive function. These include studies on various compositions of medium-chain triglycerides, lactoferrin, lactoferrin degradation products, docosahexaenoic acid (DHA), leucovorin or its pulverized form, specific amino acids, lipopolysaccharides, and other compounds and natural products. However, there are no reports establishing a link between FFAR4, or even FFAR4-expressing adipocytes, and cognitive function.

[0010] Furthermore, the use of mesenchymal stem cells in regenerative medicine to treat or improve impaired brain function is also known (Japanese Patent Application Publication No. 2019-137681 (Patent Document 6)). However, this utilizes the property of mesenchymal stem cells to differentiate into cells such as nerve cells, and does not show the correlation between FFAR4 and cognitive function.

[0011] Existing technical documents

[0012] Patent documents

[0013] Patent Document 1: Japanese Patent Application Publication No. 2012-520240

[0014] Patent Document 2: Japanese Patent Application Publication No. 2008-001690

[0015] Patent Document 3: International Publication No. 2005 / 083070

[0016] Patent Document 4: Japanese Patent Application Publication No. 2016-214135

[0017] Patent Document 5: International Publication No. 2003 / 106663

[0018] Patent Document 6: Japanese Patent Application Publication No. 2019-137681

[0019] Non-patent literature

[0020] Non-patent literature 1: Hirasawa, A. et al. Nat. Med. 11, 90-94, 2005

[0021] Non-patent literature 2: Sun Q et al. Mol. Pharmacol. 78, 804-810, 2010

[0022] Non-patent literature 3: Ichimura, A. et al. Nature 483, 350-354, 2012

[0023] Non-patent literature 4: Hara, T. et al. Rev. Physiol. Biochem. Pharmacol. 164, 77-116, 2013

[0024] Non-patent literature 5: Milligan, G. et al. Trend Pharmacol. Sci. 38, 809-821, 2017 Summary of the Invention

[0025] This invention provides a novel method for improving and even treating various diseases, such as age-related glucose intolerance and cognitive impairment.

[0026] The inventors have discovered that transplanting adipocytes that highly express FFAR4—that is, adipocytes modified to express FFAR4 by introducing the gene encoding FFAR4—into aged animals improves age-related impaired glucose tolerance. This implies that obtaining adipocytes that highly express FFAR4 could improve age-related glucose tolerance.

[0027] Furthermore, it was found that transplanting adipocytes with high FFAR4 expression into older animals improved age-related cognitive impairment. This result was entirely unexpected, as no papers had previously reported on the relationship between FFAR4 and cognitive function.

[0028] Therefore, transplanting adipocytes that highly express FFAR4 into aged animals may not only be used to treat or even prevent impaired glucose tolerance and cognitive impairment associated with aging in animals and humans, but may also be used to treat or even prevent various diseases.

[0029] Furthermore, the present invention relates to a composition for transplantation of adipocytes containing high expression of FFAR4, and further, to a method comprising transplanting a composition for transplantation of adipocytes containing high expression of FFAR4 into humans or animals for the treatment and / or prevention of various diseases, particularly for the prevention or improvement of age-related glucose intolerance and for the prevention or improvement of age-related cognitive impairment.

[0030] In another aspect, the present invention relates to a method for autologous transplantation of adipose tissue-derived stem cells collected from humans or animals, which are induced to differentiate into adipocytes by genetic modification to achieve high expression of FFAR4.

[0031] The specific embodiments of the present invention are illustrated below.

[0032] (1) An adipocyte that has been introduced with a gene encoding FFAR4 and modified to express FFAR4.

[0033] (2) With regard to the adipocytes described in item (1), the aforementioned adipocytes are cells obtained by inducing differentiation after forcibly expressing FFAR4 by introducing the FFAR4 gene into adipose stem cells or adipose precursor cells derived from adipose tissue.

[0034] (3) With regard to the adipocytes described in item (2), the aforementioned adipocytes are manufactured by a method comprising the following steps,

[0035] The process of creating a chimeric gene, wherein the aforementioned chimeric gene is a gene in which human FFAR4 cDNA (NM_181745) is positioned downstream of an appropriate promoter sequence;

[0036] b) The process of embedding the aforementioned chimeric gene into a virus or similar substance and introducing it into adipose-derived stem cells or pre-adipocytes; and

[0037] c is the process of differentiating adipose stem cells or preadipocytes into adipocytes after the aforementioned chimeric gene has been introduced.

[0038] (4) With regard to the adipocytes described in item (1), the aforementioned adipocytes are cells isolated from the adipose tissue of transgenic mice into which the gene encoding FFAR4 has been introduced.

[0039] (5) With regard to the adipocytes described in item (1), for the treatment and / or prevention of various diseases.

[0040] (6) With regard to the adipocytes described in item (5), the aforementioned disease is impaired glucose tolerance associated with aging.

[0041] (7) Regarding the adipocytes described in item (5), the aforementioned disease is associated with impaired cognitive abilities due to aging.

[0042] (8) A method for manufacturing adipocytes according to item (2), comprising:

[0043] The process of creating a chimeric gene, wherein the aforementioned chimeric gene is a gene in which human FFAR4 cDNA (NM_181745) is positioned downstream of an appropriate promoter sequence;

[0044] b) The process of embedding the aforementioned chimeric gene into a virus or similar substance and introducing it into adipose-derived stem cells or pre-adipocytes; and

[0045] c is the process of differentiating adipose stem cells or preadipocytes into adipocytes after the aforementioned chimeric gene has been introduced.

[0046] (9) With regard to the method described in item (8), the aforementioned promoter sequence is the aP2 gene promoter sequence.

[0047] (10) A transplant composition comprising the adipocytes described in item (1) for the treatment and / or prevention of disease.

[0048] (11) With regard to the transplant composition described in item (10), the aforementioned disease is impaired glucose tolerance associated with aging.

[0049] (12) With regard to the transplant composition described in item (10), the aforementioned disease is cognitive impairment associated with aging.

[0050] (13) A method of treating and / or preventing disease, comprising transplanting the transplant composition of item (10) into a human.

[0051] (14) With respect to the method described in item (13), the aforementioned transplant composition contains adipocytes that induce differentiation into adipocytes after forcibly expressing FFAR4 from adipose stem cells or preadipocytes collected from the aforementioned person through gene introduction.

[0052] (15) A method of treating and / or preventing disease, comprising transplanting the transplant composition of item (10) to an animal (excluding a human).

[0053] (16) With regard to the method described in item (15), the aforementioned transplant composition contains adipocytes that induce differentiation into adipocytes after forcibly expressing FFAR4 from adipose stem cells or preadipocytes collected from the aforementioned animals through gene introduction.

[0054] (17) A transgenic mouse having adipose tissue in which a gene encoding FFAR4 has been introduced to force the expression of FFAR4.

[0055] (18) With regard to the transgenic mice described in item (17), the aforementioned transgenic mice are manufactured by a method comprising the following steps,

[0056] The process of creating a chimeric gene, wherein the aforementioned chimeric gene is a gene in which human FFAR4 cDNA (NM_181745) is positioned downstream of an appropriate promoter sequence; and

[0057] b is the process of introducing the aforementioned chimeric gene into mouse zygotes.

[0058] (19) With regard to the transgenic mouse described in item (18), the promoter sequence in the aforementioned step a is the mouse aP2 gene promoter sequence. Attached Figure Description

[0059] Figure 1The plasmid used to create transgenic mice that overexpress FFAR4 in adipose tissue is shown. Expression of the human FFAR4 gene is controlled by the promoter of the protein aP2, which is expressed in adipose tissue. Therefore, human FFAR4 expression is restricted to adipose tissue.

[0060] Figure 2 This study compares the expression of the human FFAR4 gene in various organs of a transgenic (FFAR4-TG) mouse using RT-PCR. Strong expression was observed in white adipose tissue (SAT) and visceral adipose tissue (PAT), while low expression was observed in other organs such as brown adipocytes (BAT).

[0061] Figure 3 This graph shows the results of comparing the weight gain of FFAR4-TG mice with that of normal mice. No significant weight changes were observed on either the high-fat diet (HFD) or the normal diet (ND).

[0062] Figure 4 This graph shows the results of comparing the daily food intake of FFAR4-TG mice with that of normal mice.

[0063] Figure 5 This figure shows the results of comparing the glucose tolerance of 16-week-old (4-month-old) FFAR4-TG mice with that of 16-week-old wild-type mice.

[0064] Figure 6 This graph shows the results of comparing glucose tolerance in 60-week-old (15-month-old) FFAR4-TG mice with that in 60-week-old wild-type mice. In wild-type mice, glucose tolerance deteriorates with age; 60-week-old wild-type mice show a more rapid rise in blood glucose levels and a slower decline compared to 16-week-old wild-type mice. In 60-week-old FFAR4-TG mice, approximately the same glucose tolerance as in 16-week-old wild-type mice is shown, with no age-related deterioration in glucose tolerance observed.

[0065] Figure 7 This figure compares glucose tolerance in wild-type mice transplanted with FFAR4-TG mouse cells, mice transplanted with wild-type mouse cells, and wild-type mice that did not undergo cell transplantation. The glucose tolerance of wild-type mice transplanted with FFAR4-TG mouse cells was significantly improved compared to control mice.

[0066] Figure 8 It is Figure 7 The graph is obtained by graphically representing the area at the bottom of the line graph.

[0067] Figure 9This diagram illustrates the overview of the new substance exploration experiment. First, mice were allowed free exploration to learn about the pre-prepared substance (shown as a circle in the diagram) (top section). (The learned substance becomes the "known substance.") After 6 hours, one side of the known substance was replaced with the new substance (shown as a diamond in the diagram), and the mice were allowed to explore again. The total exploration time and the exploration time for the new substance were measured.

[0068] Figure 10 The results of novel substance exploration experiments are shown in wild-type mice and FFAR4-TG mice.

[0069] Figure 11 The results of novel substance exploration experiments are shown in wild-type mice transplanted with cells from FFAR4-TG mice, mice transplanted with cells from wild-type mice, and wild-type mice that did not undergo cell transplantation.

[0070] Figure 12 The results of a glucose tolerance test are shown in mice that underwent subcutaneous transplantation of adipose-derived stem cells infected with FFAR4 virus and subsequently induced to differentiate.

[0071] Figure 13 Show Figure 12 The area under the curve of the graph.

[0072] Figure 14 This study presents the results of a novel material exploration experiment in mice that underwent subcutaneous transplantation of adipose-derived stem cells infected with FFAR4 virus and subsequently induced to differentiate. Detailed Implementation

[0073] This invention provides adipocytes that highly express FFAR4 and are useful for the treatment and / or prevention of various diseases, as well as methods for treating and / or preventing various diseases using the aforementioned cells. The adipocytes highly expressing FFAR4 in this invention are adipocytes modified to express FFAR4 by introducing a gene encoding FFAR4, for example, including cells obtained by induced differentiation after forced expression of FFAR4 through the introduction of the FFAR4 gene into adipose-derived adipose stem cells or pre-adipocyte cells, and cells isolated from the adipose tissue of transgenic mice that have been introduced with a gene encoding FFAR4.

[0074] The embodiments of the present invention will be described in detail below. However, the following description is for the purpose of easy understanding of the present invention. Therefore, the scope of the present invention is not limited to the embodiments described below. Other embodiments that can be appropriately replaced by those skilled in the art are also included within the scope of the present invention.

[0075] 1. Explanation of terminology

[0076] The following terms, frequently used in this specification, are defined and their composition is explained in detail. It should be noted that, unless otherwise specified, the following definitions are applicable to other embodiments of the invention.

[0077] In this manual, "adipose tissue" refers to a type of connective tissue that constitutes a living organism, primarily located in the subcutaneous layer. Adipose tissue mainly contains mature fat cells and has the following functions: storing energy, protecting the body from external physical shocks and temperature changes, and secreting hormones and cytokines. In this manual, "adipose tissue" may sometimes be referred to as "fat".

[0078] In this specification, "stem cell" refers to a cell that has the ability to differentiate into various types of cells and to self-renew.

[0079] In this specification, "adipose stem cells" are somatic stem cells derived from adipose tissue, which are cells that meet the following definitions (1) to (4).

[0080] Definition of adipose stem cells

[0081] (1) It originates from adipose tissue.

[0082] (2) The plastic exhibits adhesion under the culture conditions of standard culture medium.

[0083] (3) CD90, CD73 and CD105 were positive in flow cytometry.

[0084] (4) CD31 and CD45 were negative in flow cytometry.

[0085] The adipose stem cells in this invention have at least the ability to differentiate into adipocytes.

[0086] In this invention, instead of "adipose stem cells", "adipocyte precursor cells" that can differentiate into adipocytes can be used.

[0087] The "FFAR4" in this invention is one of the G protein-coupled receptors, which has been shown to be expressed in the large intestine, adipose tissue, and lungs. Its amino acid sequence is registered in GenBank as NP_859529.

[0088] 2. Adipocytes modified to express FFAR4

[0089] The "adipocytes with high FFAR4 expression" in this invention are adipocytes modified to express FFAR4 from an artificially introduced gene, and are adipocytes that express FFAR4 much more highly than normal adipocytes without gene introduction. The degree of high expression is, for example, 1.3 to 50,000 times the standard expression level, particularly 1.3 to 100 times.

[0090] The adipocytes that highly express FFAR4 in this invention can be manufactured by inducing differentiation after introducing the FFAR4 gene into adipose stem cells or pre-adipocytes derived from adipose tissue.

[0091] For example, it can be manufactured by a method that includes the following steps.

[0092] The process of creating a chimeric gene, wherein the aforementioned chimeric gene is a gene in which human FFAR4 cDNA (NM_181745) is positioned downstream of an appropriate promoter sequence;

[0093] b) The process of embedding the aforementioned chimeric gene into a virus or similar substance and introducing it into adipose-derived stem cells or pre-adipocytes; and

[0094] c is the process of differentiating adipose stem cells or preadipocytes into adipocytes after the aforementioned chimeric gene has been introduced.

[0095] Here, the term "promoter" is defined as a DNA sequence that is essential for the specific transcription of a gene and is recognized by the cellular or introduced synthetic mechanisms. Its function is to control the transcription of one or more polynucleotides located upstream of a polynucleotide sequence (which may be multiple). It is a nucleic acid fragment structurally identified by the presence of any other DNA sequence that regulates the amount of transcription from the promoter, including but not limited to binding sites of DNA-dependent RNA polymerases, transcription initiation sites, transcription factor binding sites, repressor and activating protein binding sites, and any other DNA sequence that regulates the amount of transcription from the promoter by acting directly or indirectly.

[0096] As a "suitable promoter sequence," any promoter sequence can be used as long as the aforementioned genes can be linked in an operable manner. Furthermore, "operably linked" is defined as the promoter being in the correct position and orientation relative to the nucleic acid controlling the initiation of RNA polymerase and gene expression.

[0097] Furthermore, regarding "promoters," it is not considered important simply because a specific promoter used to control the expression of a target polynucleotide sequence can direct the expression of that polynucleotide in the targeted cell. Therefore, in the case of targeting human cells, the polynucleotide sequence coding domain can, for example, be configured adjacent to or under the control of a promoter that can be expressed in human cells. Generally, such promoters can include human promoters or viral promoters.

[0098] In various embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus terminal repeat sequence, β-actin, the rat insulin promoter, and glyceraldehyde-3-phosphate dehydrogenase can be used to obtain high-level expression of the target coding sequence. Other viral promoters, mammalian cell promoters, or bacteriophage promoters known in the art can also be considered when achieving expression of the target coding sequence, provided the expression level is sufficient to meet the intended purpose. By using promoters with known characteristics, the expression level and pattern of the target protein after transfection or transformation can be optimized.

[0099] As an example of the aforementioned promoter sequence, the aP2 gene promoter sequence can be cited.

[0100] As a method for introducing chimeric genes into adipose stem cells or adipose precursor cells, gene introduction methods based on viral vectors or non-viral vectors can be used.

[0101] In this specification, "vector" refers to a construct capable of being delivered into a host cell and optionally expressing one or more target polynucleotides. Examples of vectors are not limited to these, and may include viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and specific eukaryotic cells such as production cells. Vectors are stable and self-replicating. There are no restrictions on the types of vectors that can be used. Vectors can be cloning vectors suitable for amplifying polynucleotides, gene constructs, or expression vectors embedded in several heterologous organisms. Suitable vectors include prokaryotic expression vectors (e.g., pUC18, pUC19, Bluescript and its derivatives), mpl8, mpl9, pBR322, pMB9, CoIE1, pCR1, RP4, bacteriophages, and shuttle vectors (e.g., pSA3 and pAT28), as well as viral vectors (e.g., adenovirus, adeno-associated virus, retrovirus, and lentivirus), and non-viral vectors, such as pSilencer 4.1-CMV (Ambion (registered trademark), Life Technologies). Eukaryotic expression vectors based on Corp., Carslbad, CA, US), pcDNA3, pcDNA3.1 / hygpHCMV / Zeo, pCR3.1, pEFl / His, pIND / GS, pRc / HCMV2, pSV40 / Zeo2, pTRACER-HCMV, pUB6 / V5-His, pVAXl, pZeoSV2, pCI, pSVL, pKSV-10, pBPV-l, pML2d, and pTDT1.

[0102] In addition, as a method for producing adipocytes that express FFAR4 through artificially introduced genes, examples include methods that differentiate adipocytes into adipocytes after forcibly expressing FFAR4 through gene introduction from adipose stem cells or preadipocytes collected from humans or animals, and methods that produce adipocytes by creating transgenic mice with genes encoding FFAR4.

[0103] The expression level of FFAR4 can be determined, for example, by immunological methods using anti-FFAR4 antibodies (Western blotting, etc.) or by quantitative analysis methods of its mRNA (RT-PCR, etc.).

[0104] Based on the present invention, adipocytes modified to express FFAR4 are used to treat and / or prevent various diseases, particularly to treat and / or prevent age-related glucose intolerance and age-related cognitive impairment.

[0105] 3. Transgenic mice

[0106] Transgenic mice with the gene encoding FFAR4 inserted can be produced using a method that includes the following steps:

[0107] The process of creating a chimeric gene, wherein the aforementioned chimeric gene is a gene in which human FFAR4 cDNA (NM_181745) is positioned downstream of an appropriate promoter sequence; and

[0108] b is the process of introducing the aforementioned chimeric gene into mouse zygotes.

[0109] As the aforementioned promoter sequence, the same sequence as described above can be used, for example, the aP2 gene promoter sequence.

[0110] Alternatively, the same method as that used to introduce the chimeric gene into the fertilized egg can be used to introduce the chimeric gene into adipose stem cells or adipose precursor cells.

[0111] In the method of the present invention, the diseases that are the object of treatment or prevention are mainly age-related impaired glucose tolerance and cognitive impairment, but are not limited to these.

[0112] In addition, in this specification, "treatment" refers to improving the symptoms of a patient's or test subject's disease or delaying the progression of symptoms, and "prevention" refers to preventing the onset of a patient's or test subject's disease in advance. "Patient or test subject" primarily refers to humans, but can also be animals other than humans. Examples of animals other than humans include, but are not limited to, mammals such as dogs, cats, cattle, horses, pigs, goats, sheep, monkeys (cynomolgus monkeys, rhesus monkeys, common marmosets, Japanese macaques), ferrets, rabbits, rodents (mice, rats, gerbils, guinea pigs, hamsters), and birds such as chickens and quails.

[0113] 4. Transplantation composition

[0114] A composition for transplantation containing adipocytes expressing high levels of FFAR4, adjusted to a cell concentration, for example, 0.2 × 10⁻⁶ cells / cells in a suitable medium. 7 ~2×10 7 / ml, directly or mixed with a more effective medium, preferably a solution containing extracellular matrix such as collagen, is injected into the subcutaneous tissue or adipose tissue of humans or animals, preferably into the subcutaneous tissue.

[0115] As a pharmaceutically acceptable medium, any liquid that can be administered to a patient or test subject is acceptable, without particular limitation. Examples of pharmaceutically acceptable media include, but are not limited to, water for injection, physiological saline, culture medium, 5% glucose solution, hyaluronic acid solution, Ringer's solution, lactated Ringer's solution, acetate Ringer's solution, bicarbonate Ringer's solution, BICANATE (registered trademark) injection, amino acid solution, starting solution (solution 1), dehydration rehydration solution (solution 2), maintenance infusion (solution 3), postoperative recovery solution (solution 4), Plasma-Lyte A (registered trademark), etc.

[0116] The transplant composition of the present invention may contain additives that can be applied to patients or test subjects and that can adjust the preservation stability, isotonicity, absorbability, and / or viscosity of the aforementioned transplant composition. Examples of such additives include, but are not limited to, emulsifiers, dispersants, buffers, preservatives, wetting agents, antioxidants, chelating agents, thickeners, gelling agents, and pH adjusters. Examples of such thickeners include, but are not limited to, HES, dextran, methylcellulose, xanthan gum, carboxymethyl cellulose, and hydroxypropyl cellulose. The concentration of the aforementioned additives can be arbitrarily set as long as it is safe for use on patients or test subjects.

[0117] The transplant composition of the present invention may contain any component that can be applied to a patient or test subject. Examples of such components include, but are not limited to, salts, polysaccharides (e.g., hydroxyethyl starch (HES), dextran, etc.), proteins (e.g., albumin), dimethyl sulfoxide (DMSO), amino acids, culture medium components, etc.

[0118] The pH of the transplant composition of the present invention can be near neutral, for example, pH 5.5 or higher, pH 6.0 or higher, pH 6.5 or higher, or pH 7.0 or higher. Alternatively, it can be pH 10.5 or lower, pH 9.5 or lower, pH 8.5 or lower, or pH 8.0 or lower, but is not limited to these.

[0119] The cell concentration of the transplant composition of the present invention varies depending on the method of administration and the age, weight, symptoms, etc. of the patient or test subject, but can be any cell concentration that can be administered to the patient or test subject. There is no particular limitation on the lower limit of the aforementioned cell concentration, but for example, it is 1.0 × 10⁻⁶. 5 ≥10 cells / mL, 2.0×10 5 cells / mL or higher, 4.0 × 10 5 cells / mL or higher, 6.0 × 10 5 cells / mL or higher, 8.0 × 10 5 cells / mL or higher, 1.0 × 10 6 cells / mL or higher, 2.0 × 10 6 cells / mL or higher, 4.0 × 10 6 cells / mL or higher, 6.0 × 10 6 cells / mL or higher, 8.0 × 10 6 1.0 × 10⁻⁶ cells / mL or higher 7 Cells / mL or higher. There is no specific upper limit to the aforementioned cell concentration, but for example, it could be 1.0 × 10⁻⁶. 10 Below 1.0 × 10⁻⁶ cells / mL 9 Below 8.0 × 10⁻⁶ cells / mL 8 Below 6.0 × 10⁻⁶ cells / mL 8 Below 4.0 × 10⁻⁶ cells / mL 8 Below 2.0 × 10⁻⁶ cells / mL 8 Below 1.0 × 10⁻⁶ cells / mL or 1.0 × 10⁻⁶ cells / mL 8 Below 1 / mL.

[0120] The dosage for adipocytes with high FFAR4 expression is approximately 10. 2 ~10 10 The number of cells per individual, when administered to humans, is approximately 2 × 10⁻⁶. 5 ~2×10 8 cells / individual.

[0121] Regarding the frequency of administration of the transplant composition of the present invention, when administered to patients or test subjects, it is the frequency at which a curative effect on the disease can be achieved. The specific frequency of administration can be appropriately determined based on the form of administration, method of administration, and the age, weight, symptoms, etc., of the patient or test subject, but for example, once every 5 years, once every year, once every 6 months, once every 3 months, once every 8 weeks, once every 6 weeks, once every 4 weeks, once every 3 weeks, once every 2 weeks, once every week, twice a week, three times a week, four times a week, five times a week, six times a week, or seven times a week. Particularly preferred frequencies are once a year, once every 6 months, once every 3 months, once every 8 weeks, once every 6 weeks, or once every 4 weeks.

[0122] The administration period of the transplant composition of the present invention refers to the period during which a therapeutic or preventive effect can be obtained when the drug is administered to a patient or test subject. The specific administration period can be appropriately determined based on the form and method of administration, as well as the age, weight, and symptoms of the patient or test subject. However, the transplant composition of the present invention can be administered for a long period, for example, a period of ten years or several years. However, the transplant composition of the present invention has been confirmed to have an effect lasting at least 6 to 8 weeks after a single administration, so a single administration is also possible, without the need for multiple administrations.

[0123] Example

[0124] The invention is further described in detail through the following examples, but the invention is not limited to these examples.

[0125] Example 1

[0126] Glucose tolerance test and new substance exploration test in FFAR4-TG mice

[0127] 1. Production of FFAR4-TG mice

[0128] This work was commissioned to Transgenics. A chimeric gene was prepared by configuring human FFAR4 cDNA (NM_181745) with an N-terminal FLAG tag sequence downstream of the mouse aP2 gene promoter sequence. Furthermore, a polyadenylation signal was configured downstream of the human FFAR4 cDNA. The human FFAR4 cDNA was obtained by PCR as previously reported (1). The chimeric gene was administered to C57BL / 6 mouse zygotes via microinjection. The resulting mice were allowed free access to a normal diet and were fed a 12-hour light-dark cycle.

[0129] Figure 1The plasmid used to create FFAR4-TG mice is shown. Expression of the human FFAR4 gene is controlled by the promoter of the protein aP2, which is expressed in adipose tissue. Therefore, human FFAR4 expression is restricted to adipose tissue.

[0130] The expression of the human FFAR4 gene in various organs of the transgenic (FFAR4-TG) mice was compared using RT-PCR. Strong expression was observed in white adipose tissue (SAT) and visceral adipose tissue (PAT), while low expression was observed in other organs such as brown adipocytes (BAT). Figure 2 )

[0131] 2. Glucose Tolerance Test Method

[0132] Peripheral blood was obtained by shaving the tail of mice that had been fasting for 24 hours. Blood glucose concentration was measured using a One Touch Ultra (LifeScan) as fasting blood glucose concentration. Glucose was then administered intraperitoneally at a dose of 1.5 mg per 1 g body weight. Blood samples were collected at 15, 30, 60, 90, and 120 minutes later, and blood glucose concentration was measured using the same method.

[0133] 3. Experimental methods for exploring new substances

[0134] The novel substance exploration experiment was conducted using a modified method described in previous papers (2-4). Mice were allowed to freely search an open area for 5 minutes each over three days. On the fourth day, two identical substances were placed in the open area, and the mice were allowed to explore freely for 10 minutes. Each mouse was then returned to its original cage and allowed to roam freely for 6 hours. One of the two substances was then replaced with a new substance unknown to the mice, and they were again allowed to explore freely for 10 minutes. The exploration time for each substance was measured.

[0135] Specifically, firstly, the mice were allowed to freely explore and learn about known substances. Figure 9 The upper half). Six hours later, one of the known substances was replaced with the new substance, and the mice were allowed to explore again ( Figure 9 (The lower half). Then, the total exploration time and the exploration time for the new substance are measured. Figure 10 )

[0136] In a new substance exploration experiment, the cognitive and memory abilities of wild-type and FFAR4-TG mice for new substances were measured.

[0137] refer to

[0138] Hirasawa,A.et al.Free fatty acids regulate gut incretin glucagon-likepeptide-1 secretion through GPR120.Nat Med Jan; 11(1)90-94(2005)

[0139] Akkerman,S.et al.Object recognition testing:methodological considerations on exploration and discrimination measures.Behav Brain Res232,335-347(2012).

[0140] Antunes,M.&Biala,G.The novel object recognition memory:neurobiology,test procedure,and its modifications.Cogn Process 13,93-110(2012).

[0141] Leger,M.et al.Object recognition test in mice.Nat Protoc 8,2531-2537(2013).

[0142] [result]

[0143] 1. Increased body weight in FFAR4-TG mice

[0144] The weight gain of FFAR4-TG mice was compared with that of normal mice. No significant changes in weight were observed in either the high-fat diet (HFD) or the normal diet (ND). Figure 3 )

[0145] 2. Food intake of FFAR4-TG mice

[0146] The daily food intake of FFAR4-TG mice was compared with that of normal mice. No difference was observed between the two groups. Figure 4 )

[0147] 3. Glucose tolerance in FFAR4-TG mice

[0148] Glucose tolerance in 16-week-old (4-month-old) FFAR4-TG mice was compared with that in 16-week-old wild-type mice. No significant difference was observed between the two groups. Figure 5 )

[0149] Glucose tolerance in 60-week-old (15-month-old) FFAR4-TG mice was compared with that in 60-week-old wild-type mice. Wild-type mice exhibited deteriorating glucose tolerance due to advanced age; 60-week-old wild-type mice showed a more rapid rise in blood glucose levels and a slower decline compared to 16-week-old wild-type mice. In contrast, 60-week-old FFAR4-TG mice showed approximately the same glucose tolerance as 16-week-old wild-type mice, without the age-related deterioration of glucose tolerance observed.

[0150] ( Figure 6 )

[0151] 4. Novel Substance Exploration Experiment in FFAR4-TG Mice

[0152] Four-month-old wild-type mice spent more time exploring new substances than known substances, with approximately 70% of their time devoted to exploring new substances. This indicates that four-month-old wild-type mice could still remember known substances after 6 hours. On the other hand, 15-month-old wild-type mice, due to age-related cognitive impairment, did not remember known substances after 6 hours, so both types of mice spent roughly the same amount of time exploring substances, but the time spent exploring new substances decreased by about 50%. In contrast, FFAR4-TG mice, even at 15 months of age, remembered known substances, and their time spent exploring new substances did not decrease. This demonstrates that the cognitive abilities of FFAR4-TG mice are maintained even in old age. Figure 10 )

[0153] Example 2

[0154] Glucose tolerance test and novel substance exploration test in mice with FFAR4-overexpressing adipocyte transplantation

[0155] 1. Cell transplantation

[0156] Subcutaneous adipose tissue was collected from 16-month-old wild-type C57BL / 6 mice and FFAR4-TG mice, and connective tissue and lymph nodes were removed. After tissue fragmentation, the tissue was treated with Accumax (Funakoshi) enzyme at 37°C for 1 hour. The cell dispersion after passing through a 100 μm mesh was centrifuged to obtain a precipitate. The precipitate was washed three times with PBS and resuspended in a solution containing 153 mM NH4Cl, 10 mM NaHCO3, and 0.1 mM EDTA, and incubated at room temperature for 10 minutes. After washing the cells twice more with PBS, they were cultured in DMEM / F12 medium containing 10% fetal bovine serum, 2 mM IL-Alanyl-L-glutamate (Nakarai, Japan), and 1% penicillin / streptomycin for 6 hours. The adhered cells were then cultured for another 5 days. On day 6 of the initial culture, the culture medium was replaced with DMEM / F12 containing 2% fetal bovine serum, 2 mM IL-Alanyl-L-glutamate (Nakarai, Japan), 0.5 mM IBMX, 5 μM dexamethasone, 10 μM insulin, 200 μM indomethacin, and 1% penicillin / streptomycin, and cultured for another 2 days. Cells were recovered using acutase (Funakoshi), suspended in Matrigel, and cultured at 2 × 10⁻⁶ cells / year. 6 Cells / mouse were administered subcutaneously to 15-month-old mice.

[0157] 2. Glucose tolerance test in mice with FFAR4-overexpressing adipocyte transplantation

[0158] Stem cells were isolated and cultured from adipose tissue of 16-month-old FFAR4-TG mice and wild-type mice. Differentiation into adipocytes was induced for 2 days. The cells were then suspended in matrix gel at a concentration of 2 × 10⁶ cells per mouse. 6 Cells were transplanted subcutaneously into 15-month-old wild-type mice. Glucose tolerance was measured 8 weeks after transplantation.

[0159] Mice transplanted with wild-type mouse cells showed no change in glucose tolerance compared to age-matched wild-type mice that did not undergo cell transplantation. Wild-type mice transplanted with FFAR4-TG mouse cells showed significantly improved glucose tolerance compared to control mice. Figure 7 )

[0160] Figure 8 The bar chart is to Figure 7 The graph is obtained by graphically representing the area under the curve (AUC) of a line graph.

[0161] Figure 7 as well as Figure 8 The results showed that transplantation of adipocytes with high FFAR4 expression could prevent or even improve age-related glucose intolerance.

[0162] 3. A novel substance exploration experiment in mice with FFAR4-overexpressing adipocyte transplantation.

[0163] For older wild-type mice, cell transplantation of FFAR4-TG mouse-derived cells or wild-type mouse-derived cells was performed using the same method as in Example 1, Section 3, and novel substance exploration experiments were conducted on the cell-transplanted mice.

[0164] Even when wild-type mice were transplanted with cells derived from wild-type mice, the time to explore new substances was approximately 50%, similar to that of healthy, aged mice that did not undergo cell transplantation, and no improvement in cognitive ability was observed. However, transplantation of FFAR4-TG mouse-derived cells into aged wild-type mice resulted in improved cognitive abilities. Figure 11 )

[0165] The results above demonstrate that transplantation of FFAR4-overexpressing adipocytes can prevent and even improve age-related cognitive impairment.

[0166] Example 3

[0167] Glucose tolerance test and novel substance exploration test in mice infected with FFAR4 virus and induced to differentiate into adipose stem cells.

[0168] Adeno-associated virus (FFAR4 virus) encoding the FFAR4 gene and its mock virus were purchased from VectorBuilder. Sixteen-month-old male C57BL / 6 mice were purchased from Charles River.

[0169] Adipose tissue-derived stem cells obtained from the subcutaneous fat of 16-month-old male C57BL / 6 mice were cultured for 5 days and then infected with Mock virus and FFAR4 virus, respectively. One day later, the culture medium was replaced with induction differentiation medium, and the cells were cultured for 2 days. The cells were then harvested and 1×10⁶ cells were added to the culture medium. 6 Cells were transplanted subcutaneously into newly prepared 16-month-old male C57BL / 6 mice. A novel substance exploration test was conducted 6 weeks later, and a glucose tolerance test was conducted 8 weeks later.

[0170] The results of the glucose tolerance test are shown in Figure 12 , Figure 13 In addition, the results of the new substance exploration experiment will be presented to Figure 14In mice transplanted with cells infected with FFAR4 virus, significant improvements were observed in both the novel substance discovery test and the glucose tolerance test.

[0171] Industrial availability

[0172] This invention suggests that adipocytes with high FFAR4 expression can be used to treat and even prevent various diseases, particularly to treat and prevent age-related glucose intolerance and cognitive impairment.

Claims

1. The use of adipocytes in the preparation of medicaments for the treatment and / or prevention of age-related glucose intolerance and / or age-related cognitive impairment, characterized in that, The adipocytes were introduced with a gene encoding FFAR4 and modified to express FFAR4. The adipocytes are obtained by introducing the FFAR4 gene into adipose stem cells or pre-adipocyte cells derived from adipose tissue, thereby forcibly expressing FFAR4 and inducing their differentiation. The fat cells are manufactured by a method comprising the following steps: The process of creating a chimeric gene, wherein the chimeric gene is a gene containing human FFAR4 cDNA configured downstream of an appropriate promoter sequence, the NCBI accession number of the human FFAR4 cDNA being NM_181745; b) The process of embedding the chimeric gene into a virus or the like and introducing it into adipose-derived stem cells or adipose-derived progenitor cells; and c is the process of differentiating adipose stem cells or preadipocytes into adipocytes by introducing the chimeric gene.

2. The use as described in claim 1, characterized in that, The adipocytes are cells isolated from the adipose tissue of transgenic mice that have been introduced with the gene encoding FFAR4.

3. The use as described in claim 1, characterized in that, The promoter sequence is the aP2 gene promoter sequence.

4. The use of the transplant composition in the preparation of a medicament for treating and / or preventing age-related impaired glucose tolerance, characterized in that, The transplant composition contains adipocytes that have been introduced with the gene encoding FFAR4 and modified to express FFAR4. The adipocytes are obtained by introducing the FFAR4 gene into adipose stem cells or pre-adipocyte cells derived from adipose tissue, thereby forcibly expressing FFAR4 and inducing their differentiation. The fat cells are manufactured by a method comprising the following steps: The process of creating a chimeric gene, wherein the chimeric gene is a gene containing human FFAR4 cDNA configured downstream of an appropriate promoter sequence, the NCBI accession number of the human FFAR4 cDNA being NM_181745; b) The process of embedding the chimeric gene into a virus or the like and introducing it into adipose-derived stem cells or adipose-derived progenitor cells; and c is the process of differentiating adipose stem cells or preadipocytes into adipocytes by introducing the chimeric gene.

5. The use of the transplant composition in the preparation of a medicament for treating and / or preventing age-related cognitive impairment, characterized in that, The transplant composition contains adipocytes that have been introduced with the gene encoding FFAR4 and modified to express FFAR4. The adipocytes are obtained by introducing the FFAR4 gene into adipose stem cells or pre-adipocyte cells derived from adipose tissue, thereby forcibly expressing FFAR4 and inducing their differentiation. The fat cells are manufactured by a method comprising the following steps: The process of creating a chimeric gene, wherein the chimeric gene is a gene containing human FFAR4 cDNA configured downstream of an appropriate promoter sequence, the NCBI accession number of the human FFAR4 cDNA being NM_181745; b) The process of embedding the chimeric gene into a virus or the like and introducing it into adipose-derived stem cells or adipose-derived progenitor cells; and c is the process of differentiating adipose stem cells or preadipocytes into adipocytes by introducing the chimeric gene.

Images