Compositions for modulating carbohydrate metabolism and methods of assessing the state of carbohydrate metabolism in a subject

Abstract

To provide a means for regulating carbohydrate metabolism.SOLUTION: The present invention provides a composition for regulating carbohydrate metabolism comprising, as an active ingredient, an agent for controlling the amount of a D-amino acid in the body of a subject. Also provided are methods of assessing the state of carbohydrate metabolism in a subject by monitoring the amount of a D-amino acid in the body of the subject.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition for regulating carbohydrate metabolism and a method for assessing the state of carbohydrate metabolism in a subject. The present invention also relates to a method for regulating carbohydrate metabolism in a subject, and to the use of an agent for controlling the amount of D-amino acids in a living body for the manufacture of a pharmaceutical composition for regulating carbohydrate metabolism. [Background technology]

[0002] Advances in technology for identifying and analyzing chiral amino acids have led to progress in quantitative research into trace amounts of D- and L-amino acids in living organisms, including mammals. This has shed light on the existence and functions of some D-amino acids that, due to previous technological limitations, were treated as total amino acids (D-amino acids + L-amino acids) or, for convenience, as L-amino acids.

[0003] The amounts of D-amino acids in living organisms, tissues, cells, and body fluids change depending on the influence of ingestion, symbiotic bacteria, metabolism (decomposition, synthesis), transport, excretion, etc. (Non-Patent Documents 1 to 5), and characteristic chiral amino acid profiles are shown depending on diseases such as kidney disease, heart disease, and diabetes (Patent Document 1).Furthermore, it has been reported that D-amino acids are involved in the immune system in the intestinal tract (Non-Patent Document 6) and protect kidney-derived cells (Non-Patent Document 2).In addition, it has been reported that carbohydrate metabolism is involved in the biosynthesis of D-serine in nerve cells (Non-Patent Document 7).

[0004] It has been disclosed that the levels of D-alanine, D-proline, and D-aspartic acid in blood fluctuate in diabetes (Patent Documents 1 and 2). It has also been reported that D-alanine is localized in insulin-containing cells in the pancreatic islets of Langerhans and in cells containing adrenocorticotropic hormone in the anterior pituitary gland (Non-Patent Documents 8 and 9). However, the relationship between its presence and the pathology of diabetes and carbohydrate metabolism remains unclear. Glucose is the most important energy source in the body and is taken up by cells throughout the body from the blood. Normally, blood glucose levels (blood glucose levels) are maintained within a certain range by hormones derived from the pancreatic islets of Langerhans, the pituitary gland, the adrenal medulla, and the adrenal cortex, as well as the nervous system. When excess glucose is present in the blood, such as after a meal, the amount of glucose is controlled by the action of insulin, which promotes glycogen synthesis in the liver and muscles and stores it as fat in adipose tissue. On the other hand, when glucose levels drop, the glycogen stored in the liver and muscles is broken down into glucose by the action of insulin antagonist hormones such as glucagon, which is then supplied to the blood, maintaining glucose levels within a normal range. However, it was unknown how fluctuations in D-amino acid levels affect carbohydrate metabolism. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2013 / 140785 [Patent Document 2] Japanese Patent Application Laid-Open No. 2017-207490 [Patent Document 3] International Publication No. 2020 / 196436 [Non-patent literature]

[0006] [Non-Patent Document 1] Y. Miyoshi, R. Konno, J. Sasabe, K. Ueno, Y. Tojo, M. Mita, S. Aiso and K. Hamase, Alteration of intrinsic amounts of D-serine in mice lacking serine racemase and D-amino acid oxidase, Amino Acids, 43, 1919-1931 (2012). DOI: 10.1007 / s00726-012-1398-4

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[0007] In conditions where carbohydrate metabolism or transport is abnormal in the body, tissues, cells, organs, or body fluids, such as diabetes, the amount of glucose in the body becomes abnormal, which can cause various related diseases, and therefore methods for regulating glucose levels are desperately needed. [Means for solving the problem]

[0008] The inventors discovered a surprising phenomenon: D-amino acid oxidase (DAO) gene knockout mice (DAO- / - mice), which are thought to be involved in the metabolism of D-amino acids in living organisms, tissues, cells, organs, and body fluids, exhibit reduced blood glucose levels. Based on the knowledge that D-amino acid levels in DAO- / - mice and rats are increased (Non-Patent Document 1, Non-Patent Documents 10-12), the inventors came up with the idea that artificially altering the amount of D-amino acids in the body could modulate carbohydrate metabolism and alter blood glucose and insulin levels. After extensive research into this possibility, they discovered methods for modulating carbohydrate metabolism, particularly blood glucose levels, by administering D-amino acids that can increase or decrease D-amino acid levels in the body or by inhibiting metabolic enzymes, leading to the present invention. Specifically, the present invention encompasses the following:

[0009] [1] A composition for regulating carbohydrate metabolism, comprising an agent for controlling the amount of D-amino acids in a subject's living body as an active ingredient. [2] The composition according to item 1, wherein the regulation of carbohydrate metabolism is regulation of blood glucose levels. [3] The composition according to item 1 or 2, wherein the regulator is a D-amino acid or a modified or derivative thereof. [4] The composition of item 3, wherein the D-amino acid is selected from the group consisting of D-alanine, D-serine, and D-leucine. [5] The composition according to item 3 or 4, wherein the regulation of carbohydrate metabolism is improvement of carbohydrate metabolism. [6] The composition according to item 1 or 2, wherein the regulator is an agent that regulates the activity of a protein involved in the absorption, transport, distribution, metabolism, or excretion of a D-amino acid. [7] The composition of item 6, wherein the metabolism is degradation and / or synthesis. [8] The composition according to item 6 or 7, wherein the agent that regulates the activity of the protein is a regulator of gene expression of the protein. [9] The composition according to any one of items 6 to 8, wherein the control agent is selected from the group consisting of an antisense RNA or DNA molecule, an RNAi-inducing nucleic acid, a microRNA (miRNA), a ribozyme, a genome-editing nucleic acid and its expression vector, a small molecule compound, an aptamer, an antibody, an antibody fragment, and a combination thereof.

[10] The composition according to any one of items 6 to 9, wherein the protein is selected from the group consisting of D-amino acid oxidase, D-aspartate oxidase, and serine isomerase.

[11] The composition of any one of items 1 to 10, wherein the control agent is risperidone.

[12] The composition according to any one of items 1 to 11, wherein the carbohydrate metabolism is carbohydrate metabolism caused by the secretion and / or action of insulin and / or glucagon.

[13] The composition according to any one of items 1 to 12, wherein the carbohydrate metabolism is a carbohydrate metabolism expressed as insulin resistance or sensitivity, or glucose tolerance.

[14] The composition according to item 1 or 2, wherein the D-amino acid is derived from a symbiotic bacterium.

[15] The composition according to item 1 or 2, wherein the subject has a carbohydrate metabolism disorder.

[16] The composition according to Item 15, wherein the carbohydrate metabolism disorder is diabetes.

[17] The composition of item 1 or 2, wherein the subject has diabetic complications.

[18] The composition according to Item 17, wherein the diabetic complication is selected from the group consisting of retinopathy, nephropathy, neuropathy, and arteriosclerosis.

[19] The composition according to any one of items 1 to 18, which is a pharmaceutical.

[20] The composition according to any one of items 1 to 19, which is a food product.

[21] The composition according to Item 20, wherein the food is a health functional food or a dietary supplement.

[22] The composition according to Item 21, wherein the health functional food is a food for specified health uses or a food with nutrient functions.

[23] The composition according to any one of items 1 to 22, which is accompanied by improvement of blood pressure.

[0010]

[24] A composition for regulating blood pressure, comprising an agent for controlling the amount of D-amino acids in a subject's body as an active ingredient.

[25] The composition according to Item 24, wherein the regulation of blood pressure is a decrease in blood pressure.

[0011]

[26] A method for evaluating the state of carbohydrate metabolism in a subject by monitoring the amount of D-amino acids in the subject's living body.

[27] The method according to Item 26, wherein the subject has a carbohydrate metabolism disorder.

[28] The method according to Item 27, wherein the carbohydrate metabolism disorder is diabetes.

[0012]

[29] A method for regulating carbohydrate metabolism in a subject, comprising: Administering an agent for controlling the amount of D-amino acids in the body to a subject in need thereof. A method comprising:

[0013]

[30] Use of an agent for controlling the amount of D-amino acids in a living body for the manufacture of a pharmaceutical composition for regulating carbohydrate metabolism. [Effects of the Invention]

[0014] According to the present invention, by controlling the amount of D-amino acids in the body, it is possible to regulate carbohydrate metabolism, particularly the amount of glucose in the blood, and therefore it is possible to prevent, treat, or evaluate diseases in which carbohydrate metabolism is abnormal (e.g., diabetes). [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows the fasting blood insulin levels of mice with altered D-amino acid and carbohydrate metabolism (type 1 diabetes model). [Figure 2] FIG. 2 shows blood glucose levels during an insulin tolerance test in mice with altered D-amino acid and carbohydrate metabolism (type 1 diabetes model). [Figure 3]FIG. 3 shows blood glucose levels during a glucose tolerance test in mice with altered D-amino acid and carbohydrate metabolism (type 1 diabetes model). [Figure 4] FIG. 4 shows the blood glucose levels at any time in mice with altered D-amino acid and carbohydrate metabolism (type 1 diabetes model). [Figure 5] FIG. 5 shows fasting blood glucose levels in mice with altered D-amino acid and carbohydrate metabolism (type 1 diabetes model). [Figure 6] FIG. 6 shows the insulin resistance index of mice with altered D-amino acid and carbohydrate metabolism (type 1 diabetes model). [Figure 7] FIG. 7 shows the systolic blood pressure and diastolic blood pressure of mice with altered D-amino acid and carbohydrate metabolism (type 1 diabetes model). [Figure 8] FIG. 8 shows blood glucose levels during an insulin tolerance test in mice with altered carbohydrate metabolism (type 1 diabetes model) administered with a D-amino acid metabolic enzyme inhibitor. [Figure 9] FIG. 9 shows blood glucose levels during a glucose tolerance test in mice with altered carbohydrate metabolism (type 1 diabetes model) administered with a D-amino acid metabolic enzyme inhibitor. [Figure 10] FIG. 10 shows the casual blood glucose levels of mice with altered carbohydrate metabolism (type 1 diabetes model) administered with a D-amino acid metabolic enzyme inhibitor. [Figure 11] FIG. 11 shows fasting blood glucose levels in mice with altered carbohydrate metabolism (type 1 diabetes model) administered with a D-amino acid metabolic enzyme inhibitor. [Figure 12] FIG. 12 shows blood glucose levels during an insulin tolerance test in D-amino acid-administered mice with altered carbohydrate metabolism (type 1 diabetes model). [Figure 13] FIG. 13 shows fasting blood glucose levels in mice with altered carbohydrate metabolism (type 1 diabetes model) administered with D-amino acids. [Figure 14] FIG. 14 shows fasting blood glucose levels in mice with altered carbohydrate metabolism (type 1 diabetes model) administered with D-amino acids. [Figure 15]FIG. 15 shows the systolic blood pressure and diastolic blood pressure of mice with altered carbohydrate metabolism (type 1 diabetes model) administered with D-amino acids. [Figure 16] FIG. 16 shows the fasting blood insulin levels of mice with altered carbohydrate metabolism (type 1 diabetes model) administered a D-amino acid-free diet and D-amino acids. [Figure 17] FIG. 17 shows the casual blood glucose levels of mice with altered carbohydrate metabolism (type 1 diabetes model) administered a D-amino acid-free diet and D-amino acids. [Figure 18] FIG. 18 shows fasting blood glucose levels in mice with altered carbohydrate metabolism (type 1 diabetes model) administered a D-amino acid-free diet and D-amino acids. [Figure 19] FIG. 19 shows the amounts of insulin and glucagon in the pancreatic islets of mice with altered carbohydrate metabolism (type 1 diabetes model) fed a D-amino acid-free diet and administered D-amino acids. [Figure 20] FIG. 20 shows the casual blood glucose levels in mice with altered carbohydrate metabolism (type 2 diabetes model). DETAILED DESCRIPTION OF THE INVENTION

[0016] Hereinafter, embodiments for carrying out the present invention will be described, but the technical scope of the present invention is not limited to the following embodiments. Note that the prior art documents cited in this specification are incorporated herein by reference.

[0017] One embodiment of the present invention provides a composition for regulating carbohydrate metabolism, particularly blood glucose levels, comprising an agent for controlling the amount of D-amino acids in the body of a subject as an active ingredient. The present inventors discovered that carbohydrate metabolism, particularly blood glucose levels, can be regulated by increasing or decreasing the amount of D-amino acids in the body, leading to the invention.

[0018] As used herein, "controlling the amount of a D-amino acid in a living body" refers to intentionally increasing or decreasing the amount of a D-amino acid in a living body (e.g., in cells, tissues, organs, or body fluids). When a desired amount or concentration is present, it can be evaluated by appropriately monitoring the D-amino acid in a biological sample.

[0019] As used herein, the term "D-amino acid amount regulator" (also referred to as "D-amino acid amount control agent") refers to an agent that, when applied (e.g., administered), can increase or decrease the amount of a D-amino acid in a subject's living body (e.g., in cells, tissues, organs, or body fluids). As used herein, "regulating the amount of a D-amino acid in a cell" means that, by applying a D-amino acid amount control agent, the amount of a D-amino acid in a cell is increased or decreased, thereby controlling the amount of the D-amino acid within a desired range. As used herein, "regulating the amount of a D-amino acid in a tissue" means that, by applying a D-amino acid amount control agent, the amount of a D-amino acid in a tissue (e.g., renal tubules, glomeruli, etc.) is increased or decreased, thereby controlling the amount of the D-amino acid within a desired range. As used herein, "regulating the amount of a D-amino acid in an organ" means that, by applying a D-amino acid amount control agent, the amount of a D-amino acid in an organ (e.g., kidney, heart, etc.) is increased or decreased, thereby controlling the amount of the D-amino acid within a desired range. As used herein, "regulating the amount of D-amino acids in body fluids" means adjusting the amount of D-amino acids within a desired range by increasing or decreasing the amount of D-amino acids in body fluids (e.g., blood, urine, etc.) through the application of a D-amino acid amount control agent.

[0020] As used herein, the term "D-amino acid" refers to both "D-amino acids," which are stereoisomers of "L-amino acids," and glycine, which has no stereoisomers. Specifically, the term refers to glycine, D-alanine, D-histidine, D-isoleucine, D-allo-isoleucine, D-leucine, D-lysine, D-methionine, D-phenylalanine, D-threonine, D-allo-threonine, D-tryptophan, D-valine, D-arginine, D-cysteine, D-glutamine, D-proline, D-tyrosine, D-aspartic acid, D-asparagine, D-glutamic acid, and D-serine. Since D-cysteine ​​in biological samples is oxidized to D-cystine in vitro, in one embodiment of the present invention, the amount of D-cysteine ​​in a biological sample can be calculated by measuring D-cystine instead of D-cysteine.

[0021] As used herein, the term "biological sample" refers to a sample derived from a living organism, such as blood, plasma, serum, saliva, urine, ascites, amniotic fluid, lymph, semen, cerebrospinal fluid, nasal discharge, sweat, milk, tears, cells, tissues, etc., and the biological sample used in the present invention is preferably blood, plasma, or serum.

[0022] The amount of D-amino acids and / or L-amino acids can be measured by any method, such as chiral column chromatography, enzymatic methods, or immunological methods using monoclonal antibodies that distinguish optical isomers of amino acids. The amount of D-amino acids and / or L-amino acids in a sample in the present invention can be measured by any method known to those skilled in the art. For example, chromatographic and enzymatic methods (Y. Nagata et al., Clinical Science, 73 (1987), 105. Analytical Biochemistry, 150 (1985), 238., A. D'Aniello et al., Comparative Biochemistry and Physiology Part B, 66 (1980), 319. Journal of Neurochemistry, 29 (1977), 1053., A. Berneman et al., Journal of Microbial & Biochemical Technology, 2 (2010), 139., W. G. Gutheil et al., Analytical Biochemistry, 287 (2000), 196., G. Molla et al., Methods in Molecular Biology, 794 (2012), 273., T. Ito et al., Analytical Biochemistry, 371 (2007), 167.) etc.), antibody methods (T. Ohgusu et al., Analytical Biochemistry, 357 (2006), 15. etc.), gas chromatography (GC) (H. Hasegawa et al., Journal of Mass Spectrometry, 46 (2011), 502., MC Waldhier et al., Analytical and Bioanalytical Chemistry, 394 (2009), 695., A. Hashimoto, T. Nishikawa et al., FEBS Letters, 296 (1992), 33., H.Biomedical Chromatography, 15 (2001), 166. , M. Junge et al., Chirality, 19 (2007), 228., MC Waldhier et al., Journal of Chromatography A, 1218 (2011), 4537. Bruckner and A. Schieber. oxidation) and copper electrolyte electrolytes (CE) (H. Miao et al., Analytical Chemistry, 77 (2005), 7190., DL Kirschner et al., Analytical Chemistry, 79 (2007), 736., F. Kitagawa, K. Otsuka, Journal of Chromatography B, 879(2011), 3078., G. Thorsen and J. Bergquist, Journal of Chromatography B, 745(2000),389. oxidation) and high-performance chromatography (HPLC) (N. Nimura and T. Kinoshita, Journal of Chromatography, 352 (1986), 169., A. Hashimoto et al., Journal of Chromatography, 582 (1992), 1992; [ PMC free article ] [ PubMed ] 41., H. Bruckner et al., Journal of Chromatography A, 666 (1994), 259., N. Nimura et al., Analytical Biochemistry, 315 (2003), 262. C. Muller et al., Journal of Chromatography A, 1324 (2014), 109., S. Einarsson et al., Analytical Chemistry, 59 (1987), 1191., E. Okuma and H. Abe, Journal of Chromatography B, 660 (1994), 243., Y. Gogami et al., Journal of Chromatography B, 879 (2011), 3259., Y. Nagata et al., Journal of Chromatography, 575 (1992), 147., S. A. Fuchs et al., Clinical Chemistry, 54 (2008), 1443., D. Gordes et al., Amino Acids, 40 (2011), 553., D. Jin et al., Analytical Biochemistry, 269 (1999), 124., J. Z. Min et al., Journal of Chromatography B, 879 (2011), 3220., T. Sakamoto et al., Analytical and Bioanalytical Chemistry, 408 (2016), 517., W. F. Visser et al., Journal of Chromatography A, 1218 (2011), 7130., Y. Xing et al., Analytical and Bioanalytical Chemistry, 408 (2016), 141., K. Imai et al., Biomedical Chromatography, 9 (1995), 106., T. Fukushima et al., Biomedical Chromatography, 9 (1995), 10., R. J. Reischl et al., Journal of Chromatography A, 1218 (2011), 8379., R. J. Reischl and W. Lindner, Journal of Chromatography A, 1269 (2012), 262., S. Karakawa et al., Journal of Pharmaceutical and Biomedical Analysis, 115 (2015), 123., Hamase K, et al., Chromatography 39 (2018) 147-152, etc.).

[0023] The optical isomer separation and analysis system of the present invention may combine a plurality of separation and analysis steps. More specifically, the amount of D-amino acids and / or L-amino acids in a sample can be measured by using a method for analyzing optical isomers, which comprises the steps of: passing a sample containing components having optical isomers together with a first liquid as a mobile phase through a first column packing as a stationary phase to separate the components of the sample; individually retaining each of the components of the sample in a multi-loop unit; supplying each of the components of the sample individually retained in the multi-loop unit together with a second liquid as a mobile phase through a flow path to a second column packing having an optically active center as a stationary phase to separate the optical isomers contained in each of the components of the sample; and detecting the optical isomers contained in each of the components of the sample (Japanese Patent No. 4291628). In HPLC analysis, D- and L-amino acids may be derivatized in advance with fluorescent reagents such as o-phthalaldehyde (OPA) or 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), or diastereomerized using N-tert-butyloxycarbonyl-L-cysteine ​​(Boc-L-Cys) (Kenji Hamase and Kiyoshi Zaitsu, Analytical Chemistry, Vol. 53, pp. 677-690 (2004)). Alternatively, D- and L-amino acids can be measured by immunological methods using monoclonal antibodies that distinguish optical isomers of amino acids, such as monoclonal antibodies that specifically bind to D- or L-amino acids. Furthermore, when the total amount of D- and L-amino acids is used as an indicator, it is not necessary to separate and analyze the D- and L-isomers; amino acids can also be analyzed without distinguishing between D- and L-isomers. In this case, separation and quantification can be performed using enzymatic methods, antibody techniques, GC, CE, or HPLC.

[0024] As used herein, "adjustment of carbohydrate metabolism" refers to the use of a predetermined method to balance or correct abnormalities, excesses, or deficiencies in the catabolism or anabolism of nutrients (carbohydrates, etc.) in a subject. If the adjustment of carbohydrate metabolism has the desired effect, it may be evaluated by appropriately monitoring the amount of insulin, glucose, or metabolism-related markers in a biological sample, or by conducting a glucose tolerance test or insulin tolerance test. In one embodiment, the "adjustment of carbohydrate metabolism" that can be achieved by the present invention may be an improvement in carbohydrate metabolism, and preferably is the adjustment of blood glucose levels (e.g., a reduction in blood glucose levels).

[0025] It is known that an increase in blood glucose levels increases the osmotic pressure of blood, which in turn promotes water reabsorption in the kidneys and water excretion from cells, thereby increasing blood volume and body fluid volume, leading to an increase in blood pressure. Therefore, in one embodiment, the "regulation of carbohydrate metabolism" that can be achieved by the present invention can involve the regulation of blood pressure, for example, improvement of blood pressure (e.g., reduction of hypertension). In another embodiment, the present invention also provides a composition for regulating blood pressure, comprising, as an active ingredient, an agent for controlling the amount of D-amino acids in the body of a subject. As used herein, "regulating blood pressure" refers to adjusting a subject's blood pressure (systolic blood pressure and / or diastolic blood pressure) that deviates from the normal range (target blood pressure) to within or close to the normal range (target blood pressure), and includes, for example, lowering blood pressure that is generally considered to be hypertension.

[0026] Disorders of carbohydrate metabolism accompanied by changes in insulin sensitivity / resistance result in excessive or insufficient insulin secretion from the pancreas. Therefore, in one embodiment, the "regulation of carbohydrate metabolism" that can be achieved by the present invention may be regulation of insulin secretion, for example, improvement of insulin secretion, preferably improvement of hyperinsulinemia.

[0027] In this specification, the amounts of biomolecules and drugs such as D-amino acids, glucose, insulin, and proteins are expressed not only in terms of simple mass, weight, or amount of substance (mol), but also in any measurable physical quantity, such as mass, weight, amount of substance (mol) per tissue, cell, organ, or molecular unit, per volume or weight, or in a liquid such as blood or urine, concentration, specific gravity, or density.

[0028] D-amino acid dosage regulators that can be used in the present invention include drugs and foods that can increase or decrease the amount of D-amino acids in tissues, cells, organs, or body fluids by exogenous administration of D-amino acids or by adding or removing D-amino acids from foods. For example, drinking an aqueous solution containing D-amino acids can increase D-amino acid concentrations in blood and tissues (Non-Patent Document 1), while ingesting foods from which D-amino acids have been removed can decrease D-amino acid concentrations in blood. The D-amino acids used here may contain modified or derivative D-amino acids, or pharmaceutically acceptable salts thereof, as long as they can increase or decrease the amount of D-amino acids in the body. They may also contain pharmacologically acceptable carriers, diluents, or excipients, or may take the form of prodrugs. Furthermore, in addition to the D-amino acid dosage regulator, they may also contain metabolic improvers, hypoglycemic agents, etc. Drugs that can be used in the present invention can be formulated in dosage forms appropriate for their administration route. For oral administration, dosage forms such as tablets, capsules, liquids, powders, granules, chewable preparations, etc. can be designed, while for parenteral administration, dosage forms such as injections, powders, and infusion preparations can be designed. These preparations may also contain various pharmaceutical adjuvants, i.e., carriers and other auxiliary agents, such as stabilizers, preservatives, soothing agents, flavorings, corrigents, fragrances, emulsifiers, fillers, and pH adjusters, which can be incorporated within a range that does not impair the effects of the present invention. The optical purity of the D-amino acid used as a drug or raw material is preferably 50% or higher, more preferably 90% or higher; however, any optical purity can be selected within the effective range, and is not limited thereto.

[0029] In one embodiment, the D-amino acid amount regulator that can be used in the present invention may be selected from the group consisting of D-alanine, D-serine, and D-leucine, as well as modified forms and derivatives thereof.

[0030] By applying the present invention, it is possible to vary the amount of D-amino acids in a living body using any physiological mechanism, thereby adjusting carbohydrate metabolism in a subject. In one embodiment, the amount of D-amino acids in a living body can be controlled by adjusting the expression (enhancement, suppression, etc.) and / or activity (activation, inhibition, stimulation, etc.) of proteins involved in the absorption, transport, distribution, metabolism (synthesis and / or degradation), excretion, or action of D-amino acids, or of D-amino acid transporters or receptors.

[0031] Therefore, the regulator of D-amino acid amount that can be used in the present invention may directly or indirectly promote the gene expression of a protein involved in the absorption, transport, distribution, metabolism, or excretion of D-amino acids, and may be, for example, the protein or a vector that expresses it, or a factor that promotes the activity upstream of the cascade that promotes the expression of the protein or a vector that expresses it.

[0032] Furthermore, for example, the D-amino acid amount regulator that can be used in the present invention may directly or indirectly suppress the gene expression of a protein involved in the absorption, transport, distribution, metabolism, or excretion of D-amino acids, and may be selected from, for example, a small molecule compound, an aptamer, an antibody, an antibody fragment, an antisense RNA or DNA molecule, an RNAi-inducing nucleic acid, a microRNA (miRNA), a ribozyme, a genome-editing nucleic acid, and an expression vector thereof.

[0033] Herein, proteins involved in the absorption, transport, distribution, metabolism (synthesis and / or degradation), excretion, action, etc. of D-amino acids may be, for example, enzymes such as D-amino acid oxidase (DAO), D-aspartate oxidase (DDO), serine isomerase (SRR), DPP-4, etc. For example, DAO inhibitors (e.g., risperidone, chlorpromazine, sodium benzoate, etc.) can increase the amount of D-amino acids at the site of action by suppressing the oxidation of D-amino acids, and therefore can be used as agents for controlling the amount of D-amino acids in the present invention.

[0034] Furthermore, since D-amino acid transporters can increase or decrease the amount of D-amino acids at the source and destination, agents that act directly or indirectly on D-amino acid transporters can also be applied to the present invention.

[0035] Non-Patent Document 4 discloses that D-amino acid transporter proteins such as the SMCT family and ASCT family, which are expressed in the brain and kidney, can change the localization of D-amino acids by agonists / inhibitors. Because these transporters are affected by cooperation / competition through cotransporters (e.g., sodium ions) and scaffolds, the transport activity of D-amino acids can also be controlled by, for example, sodium / glucose cotransporter 2 (SGLT2) inhibitors. Therefore, although not intended to be limiting, proteins involved in the absorption, transport, distribution, metabolism (synthesis and / or degradation), excretion, or action of the above-mentioned D-amino acids may be proteins of the SMCT family or ASCT family.

[0036] Patent Document 3 discloses that angiotensin 2 receptor blockers (ARBs) change the amount of D-amino acids in the blood. Therefore, without intending to be limiting, the protein involved in the absorption, transport, distribution, metabolism (synthesis and / or degradation), excretion, action, etc. of the above-mentioned D-amino acids may be an angiotensin 2 receptor.

[0037] As used herein, the term "aptamer" refers to synthetic DNA or RNA molecules and peptide molecules capable of specifically binding to a target substance, and can be chemically synthesized in vitro in a short period of time. The aptamer used in the present invention can bind to proteins involved in, for example, the absorption, transport, distribution, metabolism, or excretion of D-amino acids, and inhibit their activity. The aptamer used in the present invention can be obtained, for example, by repeatedly selecting in vitro for binding to various molecular targets, such as small molecules, proteins, and nucleic acids, using the SELEX method (see Tuerk C., Gold L., Science, 1990, 249(4968), 505-510; Ellington AD, Szostak JW., Nature, 1990, 346(6287):818-822; U.S. Patent No. 6,867,289; U.S. Patent No. 5,567,588; and U.S. Patent No. 6,699,843).

[0038] As used herein, the term "antibody fragment" refers to a portion of a full-length antibody that retains its antigen-binding activity, and generally includes the antigen-binding domain or variable domain. Examples of antibody fragments include F(ab')2, Fab', Fab, or Fv antibody fragments (including scFv antibody fragments). Fragments obtainable by treating an antibody with a protease enzyme, followed by reduction as needed, are also included in the antibody fragment category. The antibody or antibody fragment used in the present invention may be any of human-derived antibodies, mouse-derived antibodies, rat-derived antibodies, rabbit-derived antibodies, antibodies from camelids such as llamas, and goat-derived antibodies. These antibodies or antibody fragments may be polyclonal or monoclonal antibodies, complete or truncated antibodies (e.g., F(ab')2, Fab', Fab, or Fv fragments), chimeric antibodies, humanized antibodies, or fully human antibodies.

[0039] As used herein, the term "antisense RNA or DNA molecule" refers to a molecule that has a base sequence complementary to a functional RNA (sense RNA), such as messenger RNA (mRNA), and that, by forming a duplex with the sense RNA, inhibits the synthesis of the protein that the sense RNA is intended to serve. In the present invention, antisense oligonucleotides containing antisense RNA or DNA molecules bind to the mRNA of proteins involved in the absorption, transport, distribution, metabolism, or excretion of D-amino acids, thereby inhibiting their translation into protein. This reduces the expression level of proteins involved in the absorption, transport, distribution, metabolism, or excretion of D-amino acids, thereby inhibiting their activity. Methods for synthesizing antisense RNA or DNA molecules are well known in the art and can be used in the present invention.

[0040] As used herein, "RNAi-inducing nucleic acid" refers to a polynucleotide capable of inducing RNA interference (RNAi) when introduced into a cell. It is typically an RNA, DNA, or chimeric molecule of RNA and DNA containing 19 to 30 nucleotides, preferably 19 to 25 nucleotides, and more preferably 19 to 23 nucleotides, and is optionally modified. RNAi may occur in mRNA or in RNA immediately after transcription before processing, i.e., RNA with a nucleotide sequence containing exons, introns, a 3' untranslated region, and a 5' untranslated region. RNAi methods that can be used in the present invention include (1) directly introducing short double-stranded RNA (siRNA) into cells, (2) incorporating short hairpin RNA (shRNA) into various expression vectors and introducing the vector into cells, or (3) constructing a vector that expresses siRNA by inserting a short double-stranded DNA corresponding to the siRNA between two promoters arranged in opposing directions, and then introducing the vector into cells. The RNAi-inducing nucleic acid may include siRNA, shRNA, or miRNA that enables cleavage of the RNA of the D-serine transporter protein or inhibition of its function, and these RNAi nucleic acids may be directly introduced using liposomes or the like, or may be introduced using an expression vector that induces these RNAi nucleic acids.

[0041] In one embodiment, the RNAi-inducing nucleic acid used in the present invention for a protein involved in D-amino acid absorption, transport, distribution, metabolism, or excretion may be any nucleic acid that exhibits the biological effect of inhibiting or significantly suppressing the expression of the protein involved in D-amino acid absorption, transport, distribution, metabolism, or excretion. Those skilled in the art can synthesize the nucleic acid by referring to the base sequence of the protein. For example, the nucleic acid can be chemically synthesized using an automated DNA / RNA synthesizer utilizing DNA synthesis techniques such as the solid-phase phosphoramidite method, or can be synthesized by an siRNA-related custom synthesis company (e.g., Life Technologies, Inc.). In one embodiment, the siRNA used in the present invention may be derived from its precursor, short-hairpin double-stranded RNA (shRNA), via processing by the intracellular RNase Dicer.

[0042] As used herein, "microRNA (miRNA)" refers to a single-stranded RNA molecule 21 to 25 bases long that is involved in post-transcriptional regulation of gene expression in eukaryotes. miRNAs generally recognize the 3'UTR of mRNA to suppress translation of target mRNA and thereby inhibit protein production. Therefore, miRNAs that can directly and / or indirectly reduce the expression level of D-serine transporter proteins are also included within the scope of the present invention.

[0043] As used herein, the term "ribozyme" refers to a general term for an enzymatic RNA molecule that can catalyze the specific cleavage of RNA. Some ribozymes are 400 nucleotides or larger, such as group I intron-type ribozymes and M1 RNA contained in RNase P, but there are also hammerhead and hairpin-type ribozymes that have an active domain of about 40 nucleotides (see, for example, Koizumi Makoto and Otsuka Eiko, Protein, Nucleic Acid, Enzymes, 1990, 35, 2191).

[0044] For example, the self-cleaving domain of a hammerhead ribozyme cleaves the 3' side of C15 in the sequence G13U14C15, but base pairing between U14 and A9 is considered to be important for its activity, and it has been shown that cleavage can also be achieved with A15 or U15 instead of C15 (see, for example, Koizumi, M. et al., FEBS Lett, 1988, 228, 228). By designing a ribozyme whose substrate binding site is complementary to an RNA sequence near the target site, it is possible to obtain an RNA-cleaving ribozyme that recognizes the sequence UC, UU, or UA in the target RNA like a restriction enzyme. Those skilled in the art can prepare such ribozymes by referring to the following literature: Koizumi, M. et al., FEBS Lett, 1988, 239, 285; Koizumi, M. and Otsuka, Eiko, Proteins, Nucleic Acids, and Enzymes, 1990, 35, 2191; Koizumi, M. et al., Nucl. Acids Res., 1989, 17, 7059.

[0045] Hairpin ribozymes can also be used in the present invention. These ribozymes are found, for example, in the minus strand of satellite RNA of tobacco ringspot virus (Buzayan, JM., Nature, 1986, 323, 349). It has been shown that target-specific RNA-cleaving ribozymes can also be produced from hairpin ribozymes (see, for example, Kikuchi, Y. & Sasaki, N., Nucl. Acids. Res., 1991, 19, 6751; Kikuchi, Hiroshi, Chemistry and Biology, 1992, 30, 112). By using ribozymes to specifically cleave the transcript of a gene encoding a D-serine transporter protein, the expression of the D-serine transporter protein can be inhibited.

[0046] In this specification, genome editing nucleic acid refers to a nucleic acid used to edit a desired gene in a system using a nuclease used for gene targeting. The nuclease used for gene targeting includes not only known nucleases but also new nucleases that will be used for gene targeting in the future. For example, known nucleases include CRISPR / Cas9 (Ran, FA, et al., Cell, 2013, 154, 1380-1389), TALEN (Mahfouz, M., et al., PNAS, 2011, 108, 2623-2628), ZFN (Urnov, F., et al., Nature, 2005, 435, 646-651), etc.

[0047] In one embodiment, commensal bacteria, including enterobacteria, are one of the living organism's sources of D-amino acids. By using antibiotics, intestinal regulators, oligosaccharides, probiotics, microbial transplants, fecal transplants, dysbiosis treatment, and other methods, the microflora and growth environment can be altered, resulting in an increase or decrease in D-amino acid levels in the living organism. While not intended to be limiting, as an example of probiotics, ingestion of yogurt containing 1073R-1 lactic acid bacteria is known to increase D-serine and decrease D-lysine in feces. Such lactic acid bacteria may be used as a D-amino acid level regulator in the present invention. However, foods known to contain D-amino acids, such as microbial fermentation products such as black vinegar, yogurt, cheese, and natto, as well as bacterial cells or bacterial cell extracts, contain many potential active ingredients in addition to D-amino acids. Therefore, when using the agent for controlling D-amino acid levels in the present invention as a D-amino acid level regulator, it is important to use an amount that can confirm the increase or decrease in D-amino acid levels at the site of action upon ingestion. In particular, since L-amino acids have various physiological activities that differ from D-amino acids, foods for which specifications (optical purity, amount) of active ingredients that increase or decrease the amount of D-amino acids are not set are not included in the scope of the compositions of the present invention.

[0048] In the present invention, any pharmaceutical or food product that can increase or decrease the amount of D-amino acids in tissues, cells, organs, or body fluids, regardless of the mechanism described above, can be used as a means of controlling the amount of D-amino acids in the body in the present invention.

[0049] In this specification, the term "drug" is used to include pharmaceutical products and quasi-drugs.

[0050] In this specification, "food" refers to food in general, but also includes general foods including so-called health foods, as well as health functional foods such as foods for specified health uses and foods with nutrient functions.Furthermore, dietary supplements (supplements, nutritional supplements), feed, food additives, etc. are also included in the foods of the present invention.

[0051] The composition of the present invention may be administered in a dosage form suitable for topical administration (e.g., on the skin, inhalation, enema, eye drop, ear drop, nasal administration, intravaginal administration), enteral administration, or parenteral administration (e.g., intravenous, intraarterial, transdermal, intramuscular injection), but enteral administration is preferred. Enteral administration includes oral administration, enteral tube administration, and enema administration. Enteral administration includes administration via a nasogastric tube, gastrostomy, or duodenal fistula. Enema administration includes administration using a suppository or enema. In either case, the dosage form of the drug is not particularly limited, and may be liquid or solid, and can be prepared according to the common general knowledge of those skilled in the art. The specific administration method is also not particularly limited, and can be suitably administered according to the common general knowledge of those skilled in the art.

[0052] The present invention can be applied to healthy individuals and patients diagnosed with or suspected of having abnormalities in carbohydrate metabolism or nutrition. Diseases associated with abnormal carbohydrate metabolism or nutrition include diabetes, carbohydrate metabolism disorders (e.g., glycogen storage disease, galactosemia, etc.), and hypoglycemia. Diabetes is a disease characterized by hyperglycemia due to insufficient insulin action (insulin secretion disorders, increased insulin resistance, etc.). Depending on the etiology, diabetes is classified into type 1 diabetes, type 2 diabetes, diabetes due to specific mechanisms or diseases (genetic abnormalities, pancreatic exocrine diseases, endocrine diseases, liver diseases, drug or chemical-induced diseases, infectious diseases, immune diseases, other genetic syndromes, etc.), and gestational diabetes. Type 1 diabetes commonly occurs in children and adolescents and is diagnosed by polyuria, dry mouth, polydipsia, weight loss, coma (diabetic ketoacidosis), increased blood glucose, increased HbA1c, increased glycohemoglobin, increased urinary glucose, decreased urinary C-peptide (CPR), and increased GAD antibodies. Treatment is primarily with insulin injections. Type 2 diabetes is common among middle-aged and elderly people and is diagnosed by obesity, family history, postprandial hyperglycemia, elevated HbA1c, elevated glycosylated hemoglobin, polyuria, dry mouth, polydipsia, weight loss, elevated fasting blood glucose, and elevated random blood glucose. In those who are insulin-independent, treatment is with diet and exercise, hypoglycemic drugs (α-glucosidase inhibitors, biguanides, thiazolidinediones, rapid-acting insulin secretagogues; glinides, DPP-4 inhibitors, sulfonylureas; sulfonylureas, SGLT2 inhibitors, etc.), GLP-1 receptor agonists, and insulin injections. In those who are insulin-dependent, insulin injections are used to supplement basal and boosted insulin secretion, and diet and exercise therapy are also implemented. Diabetes may also present with diabetic complications such as retinopathy, nephropathy, neuropathy, ischemic heart disease, cerebral infarction, arteriosclerosis, ulcers and gangrene (especially foot lesions), and coma, which are also treated. In addition, animals in which carbohydrate metabolism disorders have been induced through genetic modification or drugs, as well as cells, tissues, and organoids derived from living organisms or cultured from such organisms, are considered to be models that represent a state of the human carbohydrate metabolism system and can be used as targets.

[0053] In one embodiment of the present invention, an increase or decrease in the amount of D-amino acids in a subject's tissues, cells, organs, or body fluids affects carbohydrate metabolism. Therefore, measuring the amount of D-amino acids in a living body can be used as an indicator of the state of carbohydrate metabolism disorders and the effectiveness of treatments such as drugs, diet, and exercise. For example, monitoring the amount of D-amino acids in a living body and the amount of glucose in blood can aid in the diagnosis and evaluation of carbohydrate metabolism disorders, analysis of the mechanism of action of drugs, screening of their efficacy and toxicity, selection of treatment methods and drugs, and determination of dosage and duration. Since the amount of D-amino acids in body fluids is affected by other diseases such as kidney disease, values ​​corrected for D-amino acid levels using renal function markers such as creatinine or other markers may be used in analysis to distinguish between these diseases.

[0054] In the present invention, carbohydrate metabolism can be evaluated using casual blood glucose levels, fasting blood glucose levels, blood insulin levels, body weight, a glucose tolerance test (IGTT: intraperitoneal glucose tolerance test), an insulin tolerance test (ITT: insulin-tolerance test), etc.

[0055] As described above, a method for regulating carbohydrate metabolism by controlling the amount of D-amino acids in the body is extremely useful for the prevention, treatment, and diagnosis of carbohydrate metabolism disorders.

[0056] In one embodiment, the present invention also provides a method for regulating carbohydrate metabolism in a subject, the method comprising administering to a subject in need thereof an agent for controlling the amount of D-amino acids in the body.

[0057] In one embodiment, the present invention also provides use of an agent for controlling the amount of D-amino acids in a living body for the manufacture of a pharmaceutical composition for regulating carbohydrate metabolism.

[0058] In one embodiment, the present invention also provides a method for assessing the state of carbohydrate metabolism in a subject by monitoring the amount of D-amino acids in the subject's living body.

[0059] In one embodiment, the present invention provides a method for screening drugs or candidates that can control the amount of D-amino acids in a living body by measuring the amount of D-amino acids in tissues, cells, organs, and body fluids before and after administration of any drug, such as an antihypertensive drug or a drug for treating diabetic nephropathy. [Example]

[0060] The present invention will be described in more detail below with reference to examples, but these examples are not intended to limit the scope of the present invention. Unless otherwise specified, statistical analysis was performed using t-tests.

[0061] Example 1: Relationship between loss of activity of D-amino acid metabolism-related enzymes, carbohydrate metabolism, and blood glucose levels

[0062] In D-amino acid oxidase gene knockout (DAO-KO) mice and rats, the oxidation (decomposition) of D-amino acids is suppressed, resulting in an increase in the amount of neutral and basic D-amino acids in the body (Non-Patent Document 1, Non-Patent Documents 10-12). Similarly, in D-aspartate oxidase gene knockout (DDO-KO) mice, the amount of acidic amino acids in the body is increased (Non-Patent Document 13). In serine racemase gene knockout (SRR-KO) mice, the synthesis of D-serine is suppressed, resulting in a decrease in the amount of D-serine in the body (Non-Patent Document 1). Seven-week-old DAO-KO mice (n = 5-6), DDO-KO mice (n = 3-6), SRR-KO mice (n = 5), and control C57BL / 6 (B6) mice (n = 10) underwent left nephrectomy (-2 weeks: -2w) and then administered 50 mg / kg of streptozotocin (STZ), which is toxic to pancreatic β cells, to generate mice with altered D-amino acid and carbohydrate metabolism (type 1 diabetes model). Blood insulin levels were measured (-1 to 17 weeks), insulin tolerance tests were performed (-1, 2, and 15 weeks), and intraperitoneal glucose tolerance tests were performed. test) (week -1), random blood glucose measurement (week -1 to 18), fasting blood glucose measurement (week -1 to 17), insulin resistance index (HOMA-IR) analysis (week -1 to 17), and systolic and diastolic blood pressure measurement (week -1 to 18) were performed as appropriate.

[0063] Before STZ administration, blood insulin levels in the DAO-KO and DDO-KO groups were higher than those in the B6 group, indicating enhanced insulin secretion. After STZ administration, blood insulin levels decreased, but remained higher than those in B6 throughout the entire period in the DAO-KO and up to 4 weeks in the DDO-KO, demonstrating the ability to suppress impaired insulin secretion (Figure 1).

[0064] In a comparison of the insulin tolerance test (ITT) between weeks 0 and 15, blood glucose levels increased and insulin resistance increased in the B6 group, whereas blood glucose levels remained constant or decreased in the DAO-KO and DDO-KO groups, indicating relatively high insulin sensitivity (Figure 2).

[0065] In an IPGTT, the DAO-KO group had lower blood glucose levels than the B6 group, indicating improved glucose tolerance. There was no difference between the DDO-KO group and the B6 group (Figure 3).

[0066] After STZ administration, casual blood glucose levels increased, but remained lower in the DAO-KO and DDO-KO groups compared with the B6 group, while remaining higher in the SRR-KO group (Figure 4).Fasting blood glucose levels also increased after STZ administration, but remained lower in the DAO-KO and DDO-KO groups compared with the B6 group (Figure 5).These results indicate that an increase in D-amino acid levels in the body reduces blood glucose levels, and that a decrease in D-amino acid levels affects the increase in blood glucose levels.

[0067] HOMA-IR was lower in the DAO-KO and DDO-KO groups 17 weeks after STZ administration compared with the B6 group, indicating that the progression of insulin resistance was suppressed. (HOMA-IR = fasting blood glucose level (mg / dl) × insulin concentration (μU / mL) / 405) (Figure 6)

[0068] The systolic and diastolic blood pressures of the DAO-KO group remained lower throughout the entire study period, and those of the DDO-KO group remained lower at 14 and 18 weeks after STZ administration compared with the B6 group (control group) (Figure 7).

[0069] These results indicate that the increase or decrease in D-amino acids in the body due to the loss of activity of specific D-amino acid metabolism-related enzymes regulates carbohydrate metabolism, as manifested by insulin secretion, insulin resistance, glucose tolerance, etc., and also lowers blood pressure.

[0070] Example 2: Relationship between inhibition of D-amino acid metabolism-related enzymes, carbohydrate metabolism, and blood glucose levels

[0071] The left kidney was removed from C57BL / 6 (B6) mice (-2 weeks: -2w). One group received risperidone, a DAO inhibitor, in their drinking water (n = 3-7), while the other group received water as a control (n = 9-11). Each group was administered 50 mg / kg of streptozotocin (STZ) at week 0 to create a mouse model of altered carbohydrate metabolism. An insulin tolerance test (-1 week, 2 weeks), an intraperitoneal glucose tolerance test (-1 week), and random and fasting blood glucose measurements (-1 week, 2 weeks, and 6 weeks) were performed.

[0072] In the insulin tolerance test (ITT) before and 2 weeks after STZ administration, the risperidone group had lower blood glucose levels than the control group, indicating that insulin resistance was suppressed (Figure 8).

[0073] In an IPGTT, blood glucose levels were lower in the risperidone group compared to the control group, indicating improved glucose tolerance (Figure 9).

[0074] After STZ administration, casual blood glucose levels increased, but remained lower in the risperidone group compared to the control group (Figure 10). Furthermore, after STZ administration, fasting blood glucose levels increased, but remained lower in the risperidone group compared to the control. These results demonstrate that inhibition of the oxidation (decomposition) of D-amino acids in the body by DAO inhibitors can lower blood glucose levels (Figure 11).

[0075] These results indicate that the increase or decrease in D-amino acids in the body due to the inhibition of specific D-amino acid metabolism-related enzymes regulates carbohydrate metabolism, which is manifested by insulin resistance, glucose tolerance, etc.

[0076] Example 3: Relationship between D-amino acid administration, carbohydrate metabolism, and blood glucose level

[0077] The left kidneys of C57BL / 6 (B6) mice were removed (-2 weeks: -2w). Each mouse was given 20 mM D-alanine (n = 5-8), 80 mM D-alanine (n = 3-4), 20 mM D-serine (n = 6-7), or 80 mM D-serine (n = 4) in its drinking water. A control group (n = 8) was also given water in their drinking water. Streptozotocin (STZ) was administered at 50 mg / kg (week 0) to generate a mouse model of altered carbohydrate metabolism. Insulin tolerance tests (weeks -1 and 2), random blood glucose measurements (weeks 0-18), fasting blood glucose measurements (weeks 0-16), and systolic and diastolic blood pressure measurements (weeks -1-18) were performed.

[0078] In the insulin tolerance test (ITT) before and 2 weeks after STZ administration, the D-alanine 20 mM administration group and the D-serine 20 mM administration group showed lower blood glucose levels, lower insulin resistance, and higher insulin sensitivity compared to the control group (Figure 12).

[0079] After STZ administration, casual blood glucose levels increased, but were maintained at lower levels in the D-alanine and D-serine groups in a dose-dependent manner compared to the control group (Figure 13).Fasting blood glucose levels also increased after STZ administration, but were maintained at lower levels in the D-alanine and D-serine groups in a dose-dependent manner compared to the control group (Figure 14).These results demonstrate that D-amino acid administration reduces blood glucose levels.

[0080] Ten weeks after STZ administration, the systolic blood pressure and diastolic blood pressure were lower in the D-alanine 80 mM administration group compared to B6 (FIG. 15).

[0081] These results indicate that increasing the amount of D-amino acids in the body by administering D-amino acids not only regulates carbohydrate metabolism, which is manifested by insulin resistance, but also lowers blood pressure.

[0082] In the 80 mM D-serine group, some animals died by the 18th week, so it is important to set the dose taking into account the toxicity of D-serine.

[0083] Example 4: Relationship between D-amino acid elimination diet, carbohydrate metabolism, and blood glucose level

[0084] C57BL / 6 (B6) mice underwent left nephrectomy (week -2: -2w) and were fed a D-amino acid-free diet (D-fd). Each group received 20 mM D-alanine (n = 5), 20 mM D-serine (n = 5), 20 mM D-proline (n = 5), 20 mM D-leucine (n = 5), or water (n = 6–14) in their drinking water. A control group (n = 5) was fed a normal diet and water. Streptozotocin (STZ) was administered at 50 mg / kg (week 0) to generate mice with altered carbohydrate metabolism. Blood insulin (weeks -1 and 3), random blood glucose (weeks -1 and 2), and fasting blood glucose (weeks -1 and 3) were measured, and insulin and glucagon levels in the pancreatic islets were measured by antibody staining.

[0085] After STZ administration, blood insulin levels decreased, but the levels were lower in the D-amino acid-free diet group (D-fd) compared with the control group, indicating that STZ administration exacerbated impaired insulin secretion (Figure 16).

[0086] After STZ administration, casual blood glucose levels increased, and were higher in the D-amino acid-free diet group compared with the control group, but the increase in casual blood glucose levels was suppressed in the D-alanine, D-serine, and D-leucine groups (Figure 17). Furthermore, fasting blood glucose levels increased after STZ administration, but were higher in the D-amino acid-free diet group compared with the control group. These results indicate that the decrease in D-amino acid levels in the body due to the D-amino acid-free diet increases blood glucose levels, and that the increase in D-amino acid levels in the body due to D-amino acid administration can suppress this increase (Figure 18).

[0087] After STZ administration, the amount of insulin in the pancreatic islets of Langerhans decreased and the amount of glucagon increased in the D-amino acid deprivation group compared to the control group (FIG. 19).

[0088] These results indicate that the reduction in D-amino acid levels in the body due to a D-amino acid deprivation diet regulated carbohydrate metabolism, as indicated by the amount of insulin secreted into the blood and the amount of insulin and glucagon in the pancreatic islets of Langerhans.

[0089] Example 5: Relationship between non-insulin-dependent diabetes model, carbohydrate metabolism, and blood glucose level

[0090] db / db mice (a type 2 diabetes model) exhibited hyperphagia and obesity due to abnormalities in the leptin receptor. Blood glucose levels were measured over time (5-18 weeks) in 5- to 8-week-old mice in the following groups: a group in which 20 mM D-alanine was administered in drinking water (n=12), a group in which the D-amino acid oxidase gene was knocked out (DAO-KO) (n=5), and a control group (n=8).

[0091] Casual blood glucose levels remained lower in both the D-alanine administration group and the DAO-KO group compared to the control group, but the DAO-KO group showed an even lower value (FIG. 20).

[0092] These results indicate that the administration of D-amino acids and the increase in D-amino acids in the body due to the loss of activity of specific D-amino acid metabolism-related enzymes regulated carbohydrate metabolism, as indicated by blood glucose levels.

Claims

1. A composition for regulating carbohydrate metabolism, comprising as an active ingredient an agent for controlling the amount of D-amino acids in a target organism, wherein the agent is an agent that modulates the activity of proteins related to the absorption, transport, distribution, metabolism, or excretion of D-amino acids.

2. The composition according to claim 1, wherein the metabolism is decomposition and / or synthesis.

3. The composition according to claim 1 or 2, wherein the agent that modulates the activity of the protein is a gene expression regulator of the protein.

4. The composition according to any one of claims 1 to 3, wherein the control agent is selected from the group consisting of antisense RNA or DNA molecules, RNAi-inducible nucleic acids, microRNA (miRNA), ribozymes, genome-edited nucleic acids and their expression vectors, small molecule compounds, aptamers, antibodies, antibody fragments, and combinations thereof.

5. The composition according to any one of claims 1 to 4, wherein the protein is selected from the group consisting of D-amino acid oxidase, D-aspartate oxidase, and serine isomerase.

6. The composition according to any one of claims 1 to 4, wherein the control agent is risperidone.

7. A composition for regulating carbohydrate metabolism, comprising as an active ingredient an agent for controlling the amount of D-amino acids in a target organism, wherein the carbohydrate metabolism is carbohydrate metabolism mediated by the secretion and / or action of insulin and / or glucagon.

8. The composition according to claim 7, wherein the carbohydrate metabolism is represented by insulin resistance or sensitivity, or glucose tolerance.

9. A composition for regulating carbohydrate metabolism, comprising as an active ingredient an agent for controlling the amount of D-amino acids in a target organism, wherein the carbohydrate metabolism is represented by insulin resistance or sensitivity, or glucose tolerance.

10. The composition according to any one of claims 1 to 9, wherein the adjustment of carbohydrate metabolism is the adjustment of blood glucose levels.

11. The composition according to any one of claims 1 to 10, wherein the control agent is a D-amino acid or a modified or derivative thereof.

12. The composition according to claim 11, wherein the D-amino acid is selected from the group consisting of D-alanine, D-serine, and D-leucine.

13. The composition according to claim 11 or 12, wherein the D-amino acid is derived from symbiotic bacteria.

14. The composition according to any one of claims 1 to 8, wherein the adjustment of carbohydrate metabolism is an improvement of carbohydrate metabolism.

15. The composition according to any one of claims 1 to 14, wherein the subject is a subject having a carbohydrate metabolism disorder.

16. The composition according to claim 15, wherein the carbohydrate metabolism disorder is diabetes mellitus.

17. The composition according to any one of claims 1 to 14, wherein the subject is a subject having diabetic complications.

18. The composition according to claim 17, wherein the diabetic complications are selected from the group consisting of retinopathy, nephropathy, neuropathy, and arteriosclerosis.

19. A composition according to any one of claims 1 to 18, which is a pharmaceutical product.

20. A food product, the composition according to any one of claims 1 to 19.

21. The composition according to claim 20, wherein the food is a health functional food or a dietary supplement.

22. The composition according to claim 21, wherein the health functional food is a food for specified health uses or a nutrient functional food.

23. A composition according to any one of claims 1 to 22, which is accompanied by an improvement in blood pressure.

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