Method for evaluating agent for treating glucose metabolism disorder using silkworms and application of the same

Abstract

To provide a simple and low-cost glucose metabolism evaluation system.SOLUTION: There is provided a method for determining whether a test material is a candidate material for treating glucose metabolism disorders, the method including: (i) causing a silkworm to take feed including sucrose in the concentration of 11%(w / w) to 20%(w / w), both inclusive of the total feed to make the silkworm in the state of growth impairment caused by glucose metabolism disorders; (ii) injecting a test material into the silkworm; and (iii) measuring the weight of the silkworm injected with the test material. There is also provided a method for screening a candidate material for treating glucose metabolism disorders, the method further including (iv) selecting a candidate material that can treat glucose metabolism disorders from the test material on the basis of the weight of the silkworm. There is furthermore provided a method for fabricating a non-human model animal with glucose metabolism disorders, the method including the step of causing the silkworm to take feed for at least 30 hours, which includes sucrose in the concentration of 11%(w / w) to 20%(w / w), both inclusive of the total feed.

Description

[Technical field]

[0001] The present invention relates to a method for evaluating an agent for treating a sugar metabolism disorder using silkworms and applications thereof. [Background technology]

[0002] The number of people with diabetes worldwide is expected to reach 536.6 million (10.5% of adults) in 2021 and 783.2 million (12.2% of adults) in 2045. In addition, the number of people with borderline diabetes (prediabetes), a high-risk state for diabetes, is estimated to reach 541 million (10.6% of adults), and the demand for medicines and functional foods that improve glucose metabolism remains high. In this environment, there is a growing movement to ban or abolish animal testing using mammals, and there is an urgent need to develop alternatives to animal testing.

[0003] Development of an in vivo evaluation system using insects (silkworms), which have no animal ethics issues, is underway. Among insects, silkworms have a strong advantage as an alternative animal, since their drug pharmacokinetics and sugar metabolism are similar to those of humans, their large body size allows for multiple administration routes such as oral administration and injection, and they have been reported to develop conditions similar to those of human sugar metabolism disorders when silkworms are fed an artificial diet high in carbohydrates (high-sugar diet).

[0004] Patent Document 1 describes a method for evaluating whether a test substance is a substance that lowers blood glucose levels in humans, which comprises: (a) a step of increasing the concentration of sugar (B) in the fat body or blood of an invertebrate by feeding the invertebrate with sugar (A); (b) a step of administering the test substance to the invertebrate obtained in step (a) in which the concentration of sugar (B) in the fat body or blood is increased; and (c) a step of measuring the concentration of sugar (B) in the fat body or blood of the invertebrate to which the test substance has been administered. It also describes a method for screening for substances that lower blood glucose levels in humans, which further comprises a step of (d) selecting, from the test substances, a substance that lowers the concentration of sugar (B) in the fat body or blood of the invertebrate. Patent Document 2 describes a screening method for diabetes treatment drugs, which comprises at least the steps of: (a) increasing the concentration of sugar (B) in the fat body or blood of an invertebrate by feeding the invertebrate a sugar (A); (b) administering a test substance to the invertebrate obtained in step (a) in which the concentration of sugar (B) in the fat body or blood of the invertebrate has been increased; (c) measuring the concentration of sugar (B) in the fat body or blood of the invertebrate to which the test substance has been administered; and (d) selecting from the test substances a substance that reduces the concentration of sugar (B) in the fat body or blood of the invertebrate. The test substance is a galactose derivative. Patent documents 1 and 2 and non-patent document 1 describe that feeding silkworms with artificial feed containing added glucose increases the sugar concentration in the hemolymph and inhibits their growth, but that the growth inhibition is relieved by administering human insulin.

[0005] Patent Document 3 describes a method for evaluating whether a test substance is a candidate substance for preventing or treating human type II diabetes, characterized by comprising: (a) a step of feeding sugar (A) to silkworms to cause them to suffer from at least one or more conditions selected from the group consisting of impaired fasting glucose, impaired glucose tolerance, insulin resistance, and dyslipidemia; (b) a step of administering the test substance to the silkworms obtained in step (a) that have been caused to suffer from at least one or more conditions selected from the group consisting of impaired fasting glucose, impaired glucose tolerance, insulin resistance, and dyslipidemia; (c) a step of fasting the silkworms to which the test substance has been administered; and (d) a step of measuring the concentration of sugar (B) in the fat body or blood of the fasted silkworms; and (e) a method for screening a substance for preventing or treating human type II diabetes, characterized by further comprising a step of selecting, from the test substances, a substance that reduces the concentration of sugar (B) in the fat body or blood of the silkworms. Patent Document 3 and Non-Patent Document 2 describe that diabetes treatment drugs (metformin, pioglitazone) exhibit a blood glucose lowering effect in a silkworm hyperglycemic model created by feeding artificial feed containing glucose.

[0006] Patent Document 4 describes a method for evaluating whether a test substance is a substance that suppresses an increase in blood glucose level in humans caused by the ingestion of sucrose by exerting α-glycosidase inhibitory activity, characterized by at least comprising: (a) a step of having a test animal ingest sucrose; (b) a step of administering the test substance simultaneously with or before or after the step (a); and (c) a step of measuring the concentration of sugar in the body fluids of the test animal to which the test substance has been administered; and (d) a method for screening, from among the test substances, a substance that suppresses an increase in blood glucose level in humans caused by the ingestion of sucrose by exerting α-glycosidase inhibitory activity, characterized by further comprising a step of selecting, from among the test substances, a substance that reduces the concentration of sugar in the body fluids of the test animal. Patent Document 5 describes a method for producing or screening a diabetes prevention / treatment agent, a blood glucose level rise suppressant, a glucose uptake inhibitor, or a blood glucose spike suppressant, which is characterized by having a step of screening types of natto using a silkworm evaluation system. Non-Patent Document 3 describes that oral administration of lactic acid bacteria YM0831 using silkworms suppresses blood glucose elevation after feeding on an artificial diet containing sucrose. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent Publication No. 2009-58500 [Patent Document 2] International Publication No. WO2010 / 004916 [Patent Document 3] Patent Publication No. 2015-210100 [Patent Document 4] International Publication No. WO2017 / 061353 [Patent Document 5] Patent Publication No. 2020-180075 [Non-patent literature]

[0008] [Non-Patent Document 1] Yasuhiko Matsumoto, Eriko Sumiya, Takuya Sugita & Kazuhisa Sekimizu. An invertebrate hyperglycemic model for the identification of anti-diabetic drugs. PLoS One. 2011 Mar 30;6(3):e18292. doi: 10.1371 / journal.pone.0018292. [Non-Patent Document 2] Yasuhiko Matsumoto, Masaki Ishii, Yohei Hayashi, Shinya Miyazaki, Takuya Sugita, Eriko Sumiya & Kazuhisa Sekimizu. Diabetic silkworms for evaluation of therapeutically effective drugs against type II diabetes. Sci Rep. 2015 May 29:5:10722. doi: 10.1038 / srep10722. [Non-Patent Document 3] Yasuhiko Matsumoto, Masaki Ishii, Setsuo Hasegawa & Kazuhisa Sekimizu. Enterococcus faecalis YM0831 suppresses sucrose-induced hyperglycemia in a silkworm model and in humans. Commun Biol. 2019 May 2:2:157. doi: 10.1038 / s42003-019-0407-5. [Summary of the Invention] [Problems to be Solved by the Invention]

[0009] However, the above-mentioned evaluation systems require techniques such as injection into silkworms, blood sampling, and blood glucose measurement, as well as reagents, equipment, etc., and are therefore cumbersome. Therefore, a simple and low-cost sugar metabolism evaluation system is desired. [Means for solving the problem]

[0010] The inventors conducted intensive research to solve the above-mentioned problems and discovered that sugar metabolism can be evaluated by measuring the weight of silkworms by raising them in a specific manner, thereby completing the present invention.

[0011] That is, the present invention relates to the following. [1] A method for evaluating whether a test substance is a candidate substance for treating a glucose metabolism disorder, comprising: (i) feeding silkworms with a diet containing sucrose at a concentration of 11% (w / w) to 20% (w / w) of the total diet, thereby causing the silkworms to suffer from a state of growth deficiency due to abnormal sugar metabolism; (ii) administering the test substance to the silkworms; and (iii) Measuring the body weight of the silkworms administered the test substance. A method comprising: [2] A method for screening a candidate substance for treating a glucose metabolism disorder, comprising: (i) feeding silkworms with a diet containing sucrose at a concentration of 11% (w / w) to 20% (w / w) of the total diet, thereby causing the silkworms to suffer from a state of growth deficiency due to abnormal sugar metabolism; (ii) administering the test substance to the silkworms; (iii) measuring the body weight of the silkworms administered the test substance; and (iv) selecting a candidate substance capable of treating the glucose metabolism disorder from the test substances based on the body weight of the silkworms; A method comprising: [3] (i) The method described in [1] or [2], which comprises feeding the silkworms with feed containing sucrose at a concentration of 11% (w / w) or more and 20% (w / w) or less of the total feed for more than 30 hours. [4] (ii) The method according to any one of [1] to [3], wherein the test substance is administered orally. [5] The method according to any one of [1] to [4], comprising carrying out (i) and (ii) simultaneously. [6] The method according to any one of [1] to [5], wherein the state of growth deficiency due to abnormal glucose metabolism is manifested in silkworms as one or more of the following symptoms, compared to when the silkworms are fed a feed containing no added sucrose: A decrease in one or more of body weight and body size, an increase in the concentration of one or more selected from glucose and trehalose in the hemolymph; Increased concentration of bombyxin in the hemolymph, Insulin resistance, Decreased expression or activity of AMP-activated protein kinase (AMPK). [7] A method for producing a non-human model animal of glucose metabolism disorder, comprising feeding silkworms a diet containing sucrose at a concentration of 11% (w / w) or more and 20% (w / w) or less of the total diet for more than 30 hours. Effect of the Invention

[0012] According to the present invention, a simple and low-cost system for evaluating agents for treating abnormal glucose metabolism can be provided.

[0013] Furthermore, some aspects of the present invention provide an evaluation system that is effective for evaluating or screening test substances that have an insulin secretion promoting effect. [Brief description of the drawings]

[0014] [Figure 1]Figure 1 shows the results of investigating the occurrence of hyperglycemia and growth suppression in silkworms fed a high-sugar diet. Figure 1A: Schedule of feeding, blood lymph sampling, food intake, and body weight measurement. Figure 1B: Food intake when fed a normal diet (ND), a glucose-added diet (GD), or a sucrose-added diet (SD) for 72 hours. Figures 1C-1E: The occurrence of hyperglycemia and growth suppression in silkworms fed a glucose-added diet (GD). Figure 1C: Body weight gain, Figure 1D: Blood trehalose concentration, Figure 1E: Blood glucose concentration. Figures 1F-1H: The occurrence of hyperglycemia and growth suppression in silkworms fed a sucrose-added diet (SD). Figure 1F: Body weight gain, Figure 1G: Blood trehalose concentration, Figure 1H: Blood glucose concentration. ND: Normal diet (Silkmate PM). [Diagram 2] Figure 2 shows the results of investigating the mechanism of growth deficiency in silkworms reared on 15% SD. All figures show the results 48 hours after the start of rearing on 15% SD. Figure 2A: Weight gain and body size of silkworms. Figure 2B: Blood glucose concentration, Figure 2C: Blood bombyxin concentration. Figures 2D-H show the results of Western blotting analysis of the activation state of insulin signaling proteins. Figure 2E: phosphorylated mTOR (p-mTOR), Figure 2F: phosphorylated AKT (p-AKT), Figure 2G: phosphorylated AMPK (p-AMPK), Figure 2H: phosphorylated 4E-BP (p-4E-BP). [Diagram 3]Figure 3 shows the results of examining conditions for creating a silkworm model of growth deficiency due to abnormal sugar metabolism (hereafter, silkworm model) by feeding silkworms with diets containing different types and concentrations of added sugar, and using the effects of antidiabetic drugs under each dietary condition as an index. Figure 3A: Schedule of feeding, administration of antidiabetic drugs, weight measurement, and blood lymph collection. Figures 3B-M show the weight gain, blood trehalose concentration, and blood glucose concentration of silkworms every 3 hours up to the 12th hour when the silkworms were fed each pattern of high sugar diet for 48 hours, switched to ND from the 48th hour (0 hr), and each antidiabetic drug was administered into the blood lymph. Figures 3B-E: weight gain, Figures 3F-I: blood trehalose concentration, Figures 3J-M: blood glucose concentration. Figures 3B, F, J: 15% SD feeding, Figures 3C, G, K: 15% GD feeding, Figures 3D, H, L: 20% SD feeding, Figures 3E, I, M: 20% GD feeding. PG: pioglitazone, MT: metformin, INS: insulin. [Figure 4] Figure 4 shows the results of examining sample administration conditions for silkworms. Figure 4A: Silkworms were fed 15% SD for 48 hours, then switched to ND from the 48th hour, at the same time a drug was administered into the hemolymph, and body weight was measured 12 hours later. Figure 4B: Silkworms were fed 15% SD for 48 hours, then mixed a drug into ND from the 48th hour, and body weight was measured 12 hours later. Figure 4C: Silkworms were fed a drug mixed into 15% SD, and body weight was measured 48 hours later. [Diagram 5] Figure 5 shows the results of analyzing the changes in expression of insulin signaling proteins in silkworm fat bodies following administration of antidiabetic drugs. Figures 5A-D: Changes in the expression of p-mTOR (Figure 5B), p-AKT (Figure 5C), or p-4E-BP (Figure 5D) following administration of INS. The test schedule was as shown in Figure 4A, and fat bodies were collected 6 hours after insulin administration. Figures 5E-I: Changes in the expression of p-mTOR (Figure 5F), p-AKT (Figure 5G), p-AMPK (Figure 5H), or p-4E-BP (Figure 5I) following administration of MT or PG. The test schedule was as shown in Figure 4C, and fat bodies were collected 48 hours after feeding. [Figure 6]Figure 6 shows the results of an investigation into the effect of administration of AKT, AMPK, or PPARγ activators on weight gain. Figure 6A: Schedule of feeding, administration of each activator, weight measurement, fat body collection, and blood lymph collection. Figure 6B: Weight gain of silkworms. Figures 6C-G: Results of an analysis of changes in expression of insulin signaling proteins in silkworm fat bodies following administration of each activator. Figure 6D: p-mTOR, Figure 6E: p-AKT, Figure 6F: p-AMPK, Figure 6G: p-4E-BP. The fat bodies used for protein expression analysis were collected from silkworms in the concentration group that showed the greatest weight gain for each drug. [Figure 7] Figure 7 shows the results of examining the effects of various diabetes drugs with different mechanisms of action on a silkworm model. Figure 7A: Schedule of feeding, administration of diabetes drugs, weight measurement, fat body collection, and hemolymph collection. Figure 7B: Weight gain of silkworms by administration of various concentrations of various type 2 diabetes drugs (GC: glibenclamide, NG: nateglinide, ACA: acarbose, DPP4: DPP-4 inhibitor, EG: epagliflozin). Figure 7C: Changes in bombyxin concentration in the hemolymph of silkworms by administration of the diabetes drugs shown in Figure 7B. Figures 7D-H: Changes in the expression of insulin signaling proteins in silkworm fat bodies by administration of GC or NG. Figure 7E: p-mTOR, Figure 7F: p-AKT, Figure 7G: p-AMPK, Figure 7H: p-4E-BP. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention will be described below. The features of the present invention described below can be combined in any way. In the following, when the term "in the present invention" is used, it does not refer to a specific embodiment unless otherwise specified. In addition, when referring to a numerical range in the following, "or less" can be replaced with "less than".

[0016] <Method for evaluating whether a test substance is a candidate substance for treating glucose metabolism disorders> In one aspect, the present invention relates to a method for evaluating whether a test substance is a candidate substance for the treatment of glucose metabolism disorders (hereinafter, sometimes referred to as the evaluation method of the present invention).

[0017] In the present invention, the term "abnormal glucose metabolism" refers to a state in which blood glucose level, i.e., the concentration of glucose in the blood, is higher than the standard value (hyperglycemia), and typically refers to diabetes (including type 1 diabetes, type 2 diabetes, secondary diabetes, diabetes associated with genetic abnormalities, gestational diabetes, etc.) and borderline diabetes. Hyperglycemia may include hyperglycemia during fasting, after a meal, after a glucose load, or whenever. Abnormal glucose metabolism is, for example, abnormal glucose metabolism in mammals, and preferably abnormal glucose metabolism in humans.

[0018] In the present invention, the term "treatment" includes both therapy and prevention. Furthermore, "treatment" includes not only the improvement of one or more symptoms, but also the suppression of the worsening of one or more symptoms.

[0019] In the present invention, the "test substance" is not particularly limited, and is preferably a component that can be used in medicines or foods and beverages (e.g., functional foods and foods for specified health uses), and examples thereof include any low molecular weight organic compound, protein, polypeptide, amino acid, nucleic acid, antibody, sugar, lipid, vitamin, bacterial cell, plant extract, animal extract, inorganic compound, etc. In addition, in the present invention, the test substance may be a single component or multiple components.

[0020] (i) Inducing a state of growth deficiency due to abnormal glucose metabolism in silkworms The evaluation method of the present invention includes (i) feeding the silkworms with a diet containing sucrose at a concentration of 11% (w / w) to 20% (w / w) based on the total diet, thereby causing the silkworms to suffer from a state of growth deficiency due to abnormal glucose metabolism. Advantages of using silkworms include that their pharmacokinetics and glucose metabolism are similar to those of mammals such as humans, making them suitable for evaluating test substances intended for humans; their large body size allows for the selection of multiple administration routes such as oral administration and injection; there are few animal ethics issues; and the time required for evaluation is short.

[0021] In the present invention, the silkworm is preferably a 5th instar larva, more preferably a 0-5 day 5th instar larva, and even more preferably a 0-3 day 5th instar larva. After hatching, the silkworm larvae undergo four moltings to reach the 5th instar. The silkworm variety is not particularly limited, and for example, "Kinshu Showa" can be used.

[0022] The feed to be ingested by silkworms contains sucrose at a concentration of 11% (w / w) or more, preferably 12% (w / w) or more, more preferably 13% (w / w) or more, even more preferably 14% (w / w) or more, and particularly preferably 15% (w / w) or more, based on the total feed, or contains sucrose at a concentration of 20% (w / w) or less, preferably 19% (w / w) or less, more preferably 18% (w / w) or less, even more preferably 17% (w / w) or less, and particularly preferably 16% (w / w) or less, based on the total feed. The above concentrations may be arbitrarily combined to represent a predetermined concentration range. Moreover, "sucrose is added at a predetermined concentration to the total feed" refers to the concentration of sucrose added to the feed relative to the total feed.

[0023] In the present invention, the addition of sucrose to feed is not considered to be a requirement of the invention.

[0024] As feed for silkworms, mulberry leaves (which may be crushed), artificial feed, etc. The artificial feed is not particularly limited as long as it is suitable for raising silkworms, such as one containing mulberry leaves (which may be crushed, an extract, etc.), and for example, Silkmate PM, Silkmate 2M, Silkmate L4M, Silkmate 2S, Silkmate PS, etc. manufactured by Nippon Nosan Kogyo Co., Ltd. can be used.

[0025] The period during which the silkworms are fed the sucrose-added feed may be, for example, 30 hours or more, preferably 36 hours or more, more preferably 42 hours or more, and even more preferably 48 hours or more. This period may be, for example, 120 hours or less, preferably 108 hours or less, more preferably 96 hours or less, even more preferably 84 hours or less, and particularly preferably 72 hours or less.

[0026] A state of growth deficiency due to abnormal glucose metabolism is, for example, when compared to when silkworms are fed a diet that does not contain sucrose, one or more of the following symptoms are exhibited in the silkworm: A decrease in one or more of body weight and body size, an increase in the concentration of one or more selected from glucose and trehalose in the hemolymph; Increased concentration of bombyxin in the hemolymph, Insulin resistance, Decreased expression or activity of AMP-activated protein kinase (AMPK).

[0027] In the present invention, the body size may refer to one or more of the body length and the body circumference. The body length may be, for example, the length from the head to the uropods of the silkworm, and may be measured by any method known to those skilled in the art. The body circumference may be, for example, the body circumference of any body segment of the silkworm, not including the uropods, and may be measured by any method known to those skilled in the art.

[0028] "Hemolymph" corresponds to the body fluid of silkworms. Insects including silkworms do not have blood vessels or lymphatic vessels, and blood and lymph are not differentiated as in vertebrates, so it is called "hemolymph". Silkworm tissues and organs exist in a state of floating in the hemolymph, and such a blood circulation system is called an open vascular system. In the present invention, when "blood" or "in the blood" of silkworms is mentioned, it means "hemolymph" or "in the hemolymph" of silkworms. Hemolymph can be collected from silkworms by any method known to those skilled in the art, for example, by a method of collecting leaked hemolymph from the base of the abdominal legs of silkworm larvae using a syringe needle. The concentration of glucose or trehalose in the hemolymph can be measured by any method known to those skilled in the art, for example, by a method involving an enzyme reaction specific to the target sugar.

[0029] "Bombyxin" is an insulin-like peptide hormone in silkworms, and has the effect of lowering the concentrations of trehalose and glucose in the hemolymph. The present inventors have found that feeding silkworms with feed containing added sucrose increases the bombyxin concentration in the hemolymph of the silkworms. The bombyxin concentration in the hemolymph can be measured by any method known to those skilled in the art, for example, by an immunological method using an anti-bombyxin antibody. The anti-bombyxin antibody can be produced from a hybridoma prepared using B cells obtained from an animal immunized by administering a bombyxin-derived peptide (e.g., bombyxin II-10; GIVDECCLRP (SEQ ID NO: 1)).

[0030] "Insulin resistance" can be evaluated, for example, by the attenuation of the effect of insulin (reduction in the concentration of glucose or trehalose in the hemolymph of silkworms, growth promotion, etc.) or by the decrease in the activity of one or more insulin signaling proteins. Examples of insulin signaling proteins include mTOR, AKT, 4E-BP, p70 S6 kinase, GSK-3β, etc. The activity of insulin signaling proteins can be measured by any method known to those skilled in the art, and for example, mTOR can be measured by an immunological method using an antibody specific to phosphorylated mTOR (p-mTOR). The decrease in insulin signaling activity can be, for example, in the case of mTOR and AKT, the expression of p-mTOR, which is an activated form of these, and phosphorylated AKT (p-AKT), which is a phosphorylated form of 4E-BP, which is an inactivated form of 4E-BP, which is a phosphorylated form of 4E-BP (p-4E-BP), which is a phosphorylated form of 4E-BP, which is a phosphorylated form of 4E-BP.

[0031] "AMPK activity" is preferably AMPK activity in fat pads, and can be evaluated using the expression of phosphorylated AMPK, which is an activated form of AMPK, as an index. Measurement of phosphorylated AMPK can be performed by any method known to those skilled in the art, for example, by an immunological method using an antibody specific to phosphorylated AMPK (p-AMPK). A decrease in AMPK activity may be, for example, a decrease in the expression of p-AMPK, which is an activated form of AMPK.

[0032] In another embodiment, the glycometabolic deficiency condition is characterized in that the silkworm exhibits one or more of the following: One or more of the following selected from weight and body size is smaller than the standard value; the concentration of one or more selected from glucose and trehalose in the hemolymph is greater than a reference value; The concentration of bombyxin in the hemolymph is greater than the reference value. Insulin resistance, Decreased AMPK expression or activity. Each reference value can be selected from those known to those skilled in the art from literature, etc.

[0033] (ii) administering the test substance to the silkworms; The evaluation method of the present invention includes (ii) administering a test substance to silkworms. The method of administering the test substance to silkworms is not particularly limited and can be selected depending on the type or characteristics of the test substance, and may be, for example, oral administration, administration into the hemolymph, or injection into the intestine, and is preferably oral administration.

[0034] The dose of the test substance to be administered to silkworms can be appropriately determined by a person skilled in the art depending on the type of the test substance or the purpose of the evaluation, and may be calculated, for example, by converting the dose to be administered to a mammal into the weight of the silkworm. When administering the desired dose of the test substance to silkworms, the number of administrations may be one or more times depending on the administration method or the type of the test substance.

[0035] The test substance may be administered to the silkworms (1) during the period when the silkworms are fed with a feed containing sucrose, or (2) after the silkworms are fed with a feed containing sucrose (e.g., after the silkworms are fed with a feed containing sucrose for a certain period of time, the silkworms are fed with a feed containing no sucrose). Preferably, the evaluation method of the present invention includes (i) and (ii) being performed simultaneously, i.e. (1) the test substance is administered to the silkworms during the period when the silkworms are fed with a feed containing sucrose. More preferably, the test substance is administered to the silkworms by feeding the silkworms with a feed containing the test substance.

[0036] (iii) Measuring the body weight of the silkworms administered the test substance. The evaluation method of the present invention includes (iii) measuring the body weight of the silkworm administered with the test substance. In the evaluation method of the present invention, by measuring the body weight of the silkworm administered with the test substance, it is possible to evaluate whether the test substance may be useful for treating a glucose metabolism disorder, and does not require complicated operations such as blood sampling or techniques and devices required for blood glucose measurement.

[0037] The weight of the silkworm may be measured according to a method known to those skilled in the art, and the method is not particularly limited. The test substance can be evaluated as being useful for treating glucose metabolism disorders by increasing the weight of the silkworm or suppressing weight loss compared to an arbitrary control by administration of the test substance. The arbitrary control is, in one embodiment, when the test substance is not administered, and in another embodiment, when the test substance is administered at a smaller dose. Therefore, in one embodiment, the evaluation method of the present invention includes (iii) measuring the weight of the silkworm administered with the test substance and evaluating whether the weight increases compared to when the test substance is not administered. In another embodiment, the evaluation method of the present invention includes (iii) measuring the weight of the silkworm administered with the test substance and evaluating whether the weight increases according to the dose of the test substance.

[0038] In yet another embodiment, the administration of the test substance results in a change in the body weight of the silkworm similar to that of an arbitrary positive control known to be capable of treating glucose metabolism disorders, and thus the test substance can be evaluated as being useful for treating glucose metabolism disorders. Therefore, in one embodiment, the evaluation method of the present invention includes (iii) measuring the body weight of the silkworm administered with the test substance and comparing it with the change in body weight when an arbitrary positive control is administered. The positive control is not particularly limited, and can be, for example, a biguanide drug such as metformin, or a thiazolidine derivative drug such as pioglitazone.

[0039] In one embodiment of the evaluation method of the present invention, the body size of the silkworm can be used as an evaluation index instead of the body weight of the silkworm.

[0040] According to one embodiment of the evaluation method of the present invention, it is possible to evaluate the effect of treating abnormal glucose metabolism with a component having an insulin secretion promoting effect, such as a sulfonylurea drug such as glibenclamide, or a rapid-acting insulin secretagogue (glinide drug) such as nateglinide, etc. Therefore, according to one embodiment of the evaluation method of the present invention, it is possible to evaluate whether a component having an insulin secretion promoting effect can be useful for treating abnormal glucose metabolism.

[0041] <Method of screening candidate substances for treating glucose metabolism disorders> In one aspect, the present invention relates to a method for screening a candidate substance for treating glucose metabolism disorders (hereinafter, sometimes referred to as the screening method of the present invention). The screening method of the present invention includes the same (i) to (iii) as the evaluation method of the present invention, and each of (i) to (iii) can be selected independently of the evaluation method of the present invention, as explained in the section <Evaluation method of the present invention>.

[0042] (iv) selecting a candidate substance capable of treating the glucose metabolism disorder from the test substances based on the body weight of the silkworms; The screening method of the present invention includes (iv) selecting a candidate substance capable of treating a glucose metabolic disorder from the test substances based on the body weight of the silkworm. The candidate substance capable of treating a glucose metabolic disorder is selected from test substances evaluated as being useful for treating a glucose metabolic disorder by (iii) measuring the body weight of the silkworm administered with the test substance. The test substance used in the screening method of the present invention may be one type or two or more types. In addition, when two or more types of test substances are used in the screening method of the present invention, (i) to (iv) do not need to be performed simultaneously for each test substance, and may be performed at different times.

[0043] In the screening method of the present invention, a candidate substance capable of treating a glucose metabolism disorder can be screened by measuring the body weight of silkworms administered with a test substance, and does not require complicated procedures such as blood sampling, or techniques and devices required for blood glucose measurement, etc. Furthermore, according to one embodiment of the screening method of the present invention, a component having an insulin secretion promoting effect can be screened as a candidate substance capable of treating a glucose metabolism disorder.

[0044] <Method for producing non-human animal models of glucose metabolism disorders> In one aspect, the present invention relates to a method for producing a non-human model animal for glucose metabolism disorder (hereinafter, sometimes referred to as the production method of the present invention).

[0045] The production method of the present invention includes feeding silkworms with a feed containing added sucrose. The feed fed to silkworms contains sucrose at a concentration of 11% (w / w) or more, preferably 12% (w / w) or more, more preferably 13% (w / w) or more, even more preferably 14% (w / w) or more, and particularly preferably 15% (w / w) or more relative to the total feed, and contains sucrose at a concentration of 20% (w / w) or less, preferably 19% (w / w) or less, more preferably 18% (w / w) or less, even more preferably 17% (w / w) or less, and particularly preferably 16% (w / w) or less relative to the total feed. The above concentrations may be arbitrarily combined to represent a predetermined concentration range. Moreover, "sucrose is added to the total feed at a predetermined concentration" refers to the concentration of sucrose added to the total feed.

[0046] As feed for silkworms, mulberry leaves (which may be crushed), artificial feed, etc. The artificial feed is not particularly limited as long as it is suitable for raising silkworms, such as one containing mulberry leaves (which may be crushed, an extract, etc.), and for example, Silkmate PM, Silkmate 2M, Silkmate L4M, Silkmate 2S, Silkmate PS, etc. manufactured by Nippon Nosan Kogyo Co., Ltd. can be used.

[0047] The period during which the silkworms are fed the sucrose-added feed may be, for example, 30 hours or more, preferably 36 hours or more, more preferably 42 hours or more, and even more preferably 48 hours or more. This period may be, for example, 120 hours or less, preferably 108 hours or less, more preferably 96 hours or less, even more preferably 84 hours or less, and particularly preferably 72 hours or less.

[0048] Since the non-human model animal obtained by the production method of the present invention shows findings of glucose metabolism disorders, it is considered that it can be used for various purposes. Examples of such uses include evaluation of the effects of medicines, food and drink ingredients, etc. on glucose metabolism disorders, and elucidation of the pathology of glucose metabolism disorders. Therefore, in one aspect, the present invention relates to a method of using the non-human model animal obtained by the production method of the present invention, including using the non-human model animal obtained by the production method of the present invention for the development of a treatment agent for glucose metabolism disorders or the development of a method for treating glucose metabolism disorders. In another aspect, the present invention relates to a method of using the non-human model animal obtained by the production method of the present invention for the analysis of the mechanism of glucose metabolism disorders. EXAMPLES

[0049] The present invention will be specifically described below with reference to examples, but the present invention should not be construed as being limited thereto.

[0050] [Materials and methods] Silkworm rearing The silkworms used in the test were "Kinshu Kanewa", a hybrid of four varieties. Freshly hatched ant silkworms were obtained and raised on Silkmate original species 1-3 instar M (Silkmate PM; Nosan Corporation), an artificial feed for silkworms. Plastic containers with a volume of 800 mL and multiple small holes on the side (the holes were sealed with breathable tape) were used as rearing containers, and five silkworms were raised in each container. The temperature in the rearing room was kept at 25°C to 27°C, and the humidity at 50% to 70%. In addition, a net for removing sand was placed at the bottom of the rearing container to prevent the silkworms from touching their own feces. In addition, from the 1st to 3rd instars, food was added and sand was removed when the molting was completed, and when the 4th instar molted (molting when transitioning from the 4th instar to the 5th instar), food was removed at the time of sleep, and food was given to the 5th instar from day 0 immediately after the molting.

[0051] Collection of hemolymph and fat body Blood samples were collected by inserting a 30G needle (insulin syringe; Becton, Dickinson and Company) into the base of the compound leg of the larvae to induce leakage of a small amount of hemolymph, which was then collected into a plastic tube, immediately frozen on dry ice, and stored at -20°C.

[0052] The fat bodies were harvested by putting the silkworms in a state of suspended animation with dry ice. The silkworms were then cut open from the back to remove the midgut. The exposed fat bodies were washed with PBS and wiped dry, after which all the fat bodies were scraped off with tweezers and weighed. The harvested fat bodies were frozen in liquid nitrogen and stored at -80°C.

[0053] Measurement of trehalose and glucose concentrations (blood glucose levels) in blood lymph Measurement of trehalose concentration in hemolymph: 198 μL of buffer (Trehalose Buffer; 5 mM Tris, 137 mM NaCl, 2.7 mM KCl pH 6.7) was added to 2 μL of hemolymph (100-fold dilution), incubated at 80°C for 3 min, and then returned to room temperature. 100 μL of this hemolymph solution was mixed with 100 μL of porcine trehalase solution (10 mU / mL) diluted 2-fold with Trehalose Buffer and incubated overnight at 37°C, after which the amount of glucose generated was measured using a Glucose CII Test Wako (Fujifilm Wako Pure Chemical Corporation, Tokyo, Japan). Trehalose concentration in hemolymph was calculated from a calibration curve prepared by measuring trehalose in the same manner.

[0054] Glucose CII Test Wako was also used to measure the glucose concentration in the hemolymph. To prevent browning of the hemolymph by the tyrosinase contained in the hemolymph, 2 μL of hemolymph was sampled in 96-well plates placed on dry ice. After sampling, the plates were removed from the dry ice and 200 μL of coloring solution was immediately added. After incubation at room temperature for 15 minutes, the absorbance at a wavelength of 505 nm was measured. The glucose concentration was calculated from the glucose standard curve.

[0055] Western blotting Proteins were extracted using a lysis buffer prepared by adding 0.1% SDS, Protease Inhibitor Cocktail Set I, and Phosphatase Inhibitor Cocktail Solution II to RIPA Buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40 Substitute, 0.5% Sodium Deoxycholate, pH 7.6) at a concentration of 1 / 100. 1 mL of lysis buffer was added to 50 mg of frozen fat bodies, and the fat bodies were homogenized for 30 seconds using a Polytron homogenizer. After standing on ice for 30 minutes, the lysate was centrifuged (12,000 rpm, 10 minutes, 4°C), and the protein concentration of the resulting supernatant was measured by the BCA method. Each sample was diluted with sample buffer (125 mM Tris-HCl, 4% SDS, 20% glycerol, 0.004% bromophenol blue, 50 mM DTT, pH 6.8) and water to obtain the same protein concentration. After heat treatment at 90°C for 5 minutes, SDS-PAGE was performed using a 10% polyacrylamide gel. The proteins in the gel were transferred to a PVDF membrane and reacted with an antibody against the target protein (primary antibody) and an enzyme-labeled secondary antibody against the primary antibody, and then the proteins were analyzed using SuperSignal TM The bands of the target proteins were detected using Chemiliuminescent HRP Substrates (Thermo Fisher Scientific Logo). Band quantification was performed using Image J.

[0056] Preparation of bombyxin monoclonal antibodies A synthetic peptide (Bombyxin II-10; GIVDECCLRP (SEQ ID NO: 1)) consisting of the N-terminus to 10th amino acid of bombyxin A chain was conjugated to bovine serum albumin (BSA) (2 mg / mL) and keyhole limpet hemocyanin (KLH) (2 mg / mL) using EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) (100 mg / mL) and NHS (N-hydroxysuccinimide) (0.54 mg / mL). A mixture of bombyxin (II-10)-KLH and adjuvant (Titer MAX® Gold Adjuvant) (17.5 μg / 100 μL / mouse) was subcutaneously injected twice at intervals of one month into 7-week-old male BALB / c mice. One month after the second immunization, blood was collected from the tail vein of the mice, and the antibody titer in the blood was evaluated by ELISA. After confirming an increase in the antibody titer, bombyxin (II-10)-KLH solution (17.5 μg / 100 μL / mouse) was subcutaneously injected as the final immunization, and the spleen was removed from the mouse three days later.

[0057] The hybridomas were produced by the method shown in 2. The blood samples were taken from other mice immunized in the same way, and the IgG bombyxin antibodies in the blood were labeled with HRP using the Peroxidase Labeling Kit - NH2 and used as bombyxin polyclonal antibodies.

[0057] The spleen excised in

[0056] was washed with ice-cold PBS, and then the tissue pieces were crushed and passed through a 40 μm cell strainer. The supernatant was removed by centrifugation (1500 rpm, 5 min), and the precipitate was diluted with hemolysis buffer (155 mmol / L NH 4 Cl, 10 mmol / L KHCO 3, 0.1 mmol / L EDTA) was added and allowed to stand for 5 minutes. The same volume of PBS was added to stop hemolysis, and after centrifugation, serum-free RPMI medium was added to the precipitate and resuspended. This spleen cell liquid was mixed with 1 / 10 volume of myeloma cell (P3U1) culture medium, and after centrifugation, 50% polyethylene glycol 1500 was added to the precipitate to cause cell fusion. The cell suspension was suspended in RPMI medium containing 15% FBS and 3 × 10 5 The hybridomas were seeded onto a 96-well microplate at 100 cells / mL. After that, in order to select only the hybridomas, half of the culture medium was replaced with HAT medium (15% FBS), a medium in which only hybridomas can survive, and cultured for 1 to 3 days. After confirming that only the hybridomas were alive, the medium was replaced with HT medium (15% FBS) and cultured for 7 days. After that, the medium was replaced with RPMI medium (15% FBS), and colonies were confirmed in many wells. In order to screen for bombyxin antibody-producing hybridomas,

[0058] The presence or absence of antibodies in the cell culture supernatant was confirmed by the ELISA method shown in . The cell populations in the wells in which antibody production was confirmed were separated using the limiting dilution method and each was grown in a different well to establish cells producing a single type of antibody. The selected single antibody-producing cells were cultured and grown, suspended in serum-free RPMI medium, and 200 μL of the suspension was administered intraperitoneally to mice. Note that the mice were intraperitoneally administered 200 μL of synthetic pristane one week before the administration of the cell suspension. One week later, ascites was collected, and the bombyxin (II-10) monoclonal antibody was purified using a protein G binding column (Spin column based Antibody Purification Kit (Protein G) COSMO BIO, Tokyo, Japan).

[0058] Screening for bombyxin antibody producing cells 100 μL of bombyxin (II-10)-BSA (protein concentration 2 μg / mL) was added to each well of a Nunc 96-well immunomicroplate and incubated overnight. After washing three times with PBS-T (phosphate buffer containing 0.1% Tween 20), 100 μL of hybridoma cell culture medium was added to each well and incubated for 1 hour. The wells were washed three times, and HRP-labeled bombyxin polyclonal antibody was added and incubated for 1 hour. WSE-7145 EzELISA TMB (ATTO, Tokyo, Japan) was used for the color reaction, and after the reaction, the absorbance at 450 nm was measured using a plate reader.

[0059] Measurement of bombyxin concentration The ELISA method described above was modified. 100 μL of bombyxin (II-10) monoclonal antibody (5 μg / mL) was added to a Nunc 96-well immunomicroplate and incubated overnight. After washing three times with PBS-T, 100 μL of hemolymph diluted 100-fold with PBS was added to each well and incubated for 1 hour. After washing three times, 100 μL of HRP-labeled bombyxin (II-10) polyclonal antibody was added to each well and incubated for 1 hour. Color was developed using WSE-7145 EzELISA TMB (ATTO, Tokyo, Japan), and the absorbance at 450 nm was measured. The concentration of bombyxin was calculated from the standard curve of bombyxin (II-10) measured at the same time.

[0060] [result] Creation of a silkworm model of growth deficiency caused by abnormal sugar metabolism (silkworm model) 1) Hyperglycemia and growth suppression in silkworms fed a high-sugar diet To determine the type and concentration of sugar suitable for creating a silkworm model in which the effect of the sample on improving sugar metabolism can be evaluated using body weight gain as an indicator, and to determine the test period, 5th instar silkworms on day 1 were fed artificial diet containing 10%, 15%, or 20% glucose (10GD, 15GD, 20GD) or sucrose (10SD, 15SD, 20SD), and body weight and glucose and trehalose concentrations in the hemolymph were measured every 24 hours for up to 72 hours (Fig. 1A).

[0061] Food intake decreased depending on the sugar concentration in the diet (Fig. 1B). Furthermore, growth was significantly suppressed when the sugar concentration in the diet was 15% or higher (Fig. 1C, F). In particular, growth almost stopped after 24 hours in the 20GD and 20SD groups. On the other hand, there was no significant difference in growth rate between the 10GD and 10SD groups and the control group fed a normal diet.

[0062] The glucose and trehalose concentrations in the hemolymph increased in both the GD group (Fig. 1D, E) and the SD group (Fig. 1G, H) depending on the sugar concentration in the diet. When comparing the same sugar concentration, the GD group was higher than the SD group at all blood sampling points. The trehalose concentration increased to approximately 4000 mg / dl in the 15GD and 20GD groups, whereas it increased to 2000 mg / dl in the 15SD and 20SD groups. The glucose concentration increased to approximately 600 mg / ml in the 15GD and 20GD groups, whereas it increased to approximately 300 mg / ml in the 15SD and 20SD groups. The increase in glucose concentration in the SD group was approximately half of that in the GD group of the same concentration. The pattern of increase in glucose concentration was a large increase in the first 48 hours in the GD group (Fig. 1D, E), followed by a gradual increase until 72 hours. The glucose concentration in the SD group (Fig. 1G, H) continued to increase until 72 hours in the 10SD group, but a large increase in the first 24 hours in the 15SD and 20SD groups, followed by a gradual increase until 72 hours.

[0063] Since there was no significant difference in growth between the group fed a diet with a 10% sugar concentration and the control group fed a normal diet, we determined that this group was not suitable for creating a silkworm model. To find clues to determine which of the remaining four conditions, namely 15GD, 20GD, 15SD, and 20SD, would be suitable for creating a model, we next investigated the mechanism of growth inhibition in hyperglycemic silkworms.

[0064] 2) Mechanism of growth suppression in hyperglycemic silkworms It has been reported that hyperglycemic silkworms develop insulin resistance. Since it was suggested that insufficient insulin action is one of the factors that inhibit growth, we analyzed the activation state of insulin signaling proteins in the fat body (a silkworm tissue that functions like a mammalian liver and adipose tissue) of silkworms raised on 15SD by Western blotting. In addition, to confirm whether insufficient secretion of bombyxin (a hormone equivalent to insulin in mammals) is a factor in growth inhibition, we created an original bombyxin antibody and constructed an ELISA system to measure the bombyxin concentration in the hemolymph. As shown in Figures 2A-C, in silkworms that had been fed 15SD for 48 hours, the concentration of bombyxin (Figure 2C) increased in conjunction with the increase in the sugar concentration in the hemolymph (Figure 2B). On the other hand, phosphorylated mTOR (p-mTOR) (Fig. 2E) and phosphorylated AKT (p-AKT) (Fig. 2F), which are activated forms of insulin signaling proteins mTOR and AKT, were decreased, and phosphorylated 4E-BP (p-4E-BP; inactivation promotes growth) (Fig. 2H), an inactive form of the transcription factor 4E-BP involved in growth inhibition, was also decreased (Fig. 2D-H), suggesting that hyperglycemic silkworms have a decreased activity of the insulin signaling system (insulin resistance). Furthermore, the expression level of p-AMPK (Fig. 2G), an activated form of AMPK, was also decreased in hyperglycemic silkworms. These results suggest that the expression of insulin resistance and decreased AMPK activity are factors in the growth inhibition in hyperglycemic silkworms.

[0065] 3) Examination of conditions for creating a silkworm model using the release of growth suppression caused by administration of diabetes drugs as an indicator 3-1 Examination of conditions for creating a silkworm model In hyperglycemic silkworms, the expression of insulin resistance and reduced AMPK activity were thought to be factors in the growth suppression. Therefore, we administered a diabetes drug that has the primary effect of improving these conditions to silkworms reared on 15GD, 20GD, 15SD, and 20SD and examined whether weight gain was observed.

[0066] Silkworms were raised on each feed condition for 48 hours from the first day of the fifth instar, and then the diabetes medications metformin, pioglitazone, and human insulin were injected (administered into the hemolymph). Immediately afterwards, the diet was switched to normal diet, and weight was measured and hemolymph samples were taken every three hours for up to 12 hours, and glucose and trehalose concentrations were measured (Figure 3A).

[0067] Figures 3A-D show the body weight at each time point. Among the four dietary conditions examined, the only group in which the body weight increase was greater than that of the non-treated group by 15 SD (Figure 3B) for all three drugs. The glucose concentration in the hemolymph did not differ between the antidiabetic drug-treated and non-treated groups under any condition (Figures 3F-M).

[0068] These results suggest that the conditions suitable for creating a silkworm model, in which the effects of diabetes treatment are apparent, are to administer Silkmate PM containing 15% sucrose for 48 hours.

[0069] 3-2 Optimization of sample administration conditions In order to investigate the administration conditions of samples that would more clearly show the weight gain effect in the silkworm model, metformin and pioglitazone were administered to the silkworm hyperglycemia model under the following three conditions, and growth (weight gain) was measured: (1) Silkworms on the first day of the fifth instar were fed 15SD for 48 hours, then switched to a normal diet from the 48th hour, and at the same time the drug was administered into the hemolymph, and the weight was measured 12 hours later, (2) Silkworms on the first day of the fifth instar were fed a 15SD diet for 48 hours, then fed a normal diet mixed with the drug, and the weight was measured 12 hours later, (3) Silkworms on the first day of the fifth instar were fed a 15SD diet mixed with the drug, and the weight was measured 48 hours later.

[0070] As shown in Figure 4A-C, under condition (3) (Figure 4C), the weight gain of the treatment group was the largest compared to the non-treatment group. This result suggested that the most suitable method of administering the evaluation sample was to mix it with 15SD and feed it to the mice starting from the first day of the fifth instar.

[0071] 4) Molecular verification of the efficacy of antidiabetic drugs in the silkworm model To verify that the improvement of the insulin signaling system was the cause of weight gain, the activation state of insulin signaling system proteins was analyzed by Western blotting in fat bodies collected 6 hours after insulin administration in the experiment 3-1, and in fat bodies collected from silkworms fed 15SD mixed with drugs for 48 hours from the 1st day of the 5th instar in the experiment 3-2 (the group whose body weight increased significantly compared to the control group in the experiment 3-2). In the insulin administration group, the expression of p-mTOR, p-AKT, and p-4E-BP was increased compared to the control group (Figure 5A-D). In addition, the expression of p-mTOR, p-AKT, and p-4E-BP was increased in the pioglitazone and metformin groups, and the expression of phosphorylated AMPK (p-AMPK), an activated form of AMPK, was also increased in the metformin group (Figure 5E-I). Activation of the insulin signaling pathway by pioglitazone, metformin, and insulin, and activation of AMPK by metformin are similar to those in mammals.

[0072] Since it was shown that activation of the insulin signaling pathway contributes to the release of growth inhibition in the silkworm model, the silkworm model we constructed is useful as an evaluation system for improving glucose metabolism.

[0073] 5) Examination of the effect of AKT, AMPK, and PPARγ activators on weight gain In the antidiabetic drug administration group, p-AKT, an activated form of AKT, was increased, and growth of the silkworm model was promoted accordingly. Therefore, we evaluated the weight gain when AKT activator (SC79), which specifically activates AKT, was administered to the silkworm model in the feed (Fig. 6A). We also evaluated the weight gain when AMPK activator (AICAR), which is the action site of metformin, and PPARγ activator (GW1929), which is the action site of pioglitazone, were administered to the silkworm model in the feed (Fig. 6B). The results showed that both activators increased body weight compared to the non-administered group. Western blotting of fat pads collected 48 hours after the start of the feed administration showed that the expression of p-mTOR, p-AKT, and p-4E-BP was increased in the SC79 administration group, which is an AKT activator (Fig. 6C-G). The expression of p-AMPK was significantly increased in the AICAR administration group, which is an AMPK activator. In addition, the expression of p-AKT tended to increase in the AICAR and GW1929 (PPARγ activator) groups. No significant differences were observed in blood glucose levels and hemolymph bombyxin concentrations. These results also indicate that the insulin signaling pathway and AMPK activation contribute to the release of growth suppression in the silkworm model.

[0074] Further potential for drug efficacy detection in the silkworm model In addition to improving insulin resistance, the targets of type 2 diabetes drugs include insulin secretion promotion, α-glucosidase inhibition (inhibition of glucose absorption from the digestive tract), DPP-4 inhibition (increasing the amount of incretin that promotes insulin secretion and suppresses glucagon secretion), SGLT2 inhibition (inhibition of glucose reabsorption in the kidney), GLP-1 receptor agonist (promoting insulin secretion and suppressing glucagon secretion), etc. To examine whether these effects can be detected in the silkworm hyperglycemia model, glibenclamide (GC; sulfonylurea (SU) insulin secretion promotion), nateglinide (NG; glinide drug, rapid-acting insulin secretion promoter), acarbose (ACA; α-glucosidase inhibitor), DPP-4 inhibitor (DPP-4), and epagliflozin (EG; SGLT2 inhibitor) were mixed into 15SD and the weight gain was evaluated after 48 hours of feeding (Figure 7A).

[0075] The weight gain effect was observed only in the groups administered GC and NG, which are insulin secretagogues (Figure 7B). The GC group showed a significant increase in body weight compared to the non-administered group, and the NG group showed a tendency to increase (Fig. 7B). The blood bombyxin concentration was significantly increased in the GC group and tended to increase in the NG group, but other drugs did not increase the blood bombyxin concentration (Fig. 7C). Insulin signaling-related proteins in the fat body 48 hours after the start of the test (Fig. 7D), the expression of p-AKT tended to increase in the GC and NG groups (Fig. 7F), and the expression of p-4E-BP was significantly increased (Fig. 7H). No significant difference in blood glucose level was observed in all administration groups compared to the control group. These results indicate that if growth promotion is observed in the silkworm model, it may be expressed through the promotion of insulin secretion.

[0076] These results suggest that the silkworm model is also useful for screening materials that have insulin secretion-promoting effects. [Industrial Applicability]

[0077] The evaluation method, screening method, and production method of the present invention using silkworms may be a useful alternative to animal testing for evaluating sugar metabolism as animal testing is increasingly phased out.

Claims

1. 1. A method for evaluating whether a test substance is a candidate substance for treating a glucose metabolism disorder, comprising: (i) feeding the silkworms with a feed containing sucrose at a concentration of 11% (w / w) to 20% (w / w) based on the total feed, thereby causing the silkworms to suffer from growth deficiency due to abnormal sugar metabolism; (ii) administering a test substance to the silkworm; and (iii) Measuring the body weight of the silkworms administered the test substance. A method comprising:

2. A method for screening a candidate substance for the treatment of glucose metabolism disorders, comprising: (i) feeding the silkworms with a feed containing sucrose at a concentration of 11% (w / w) to 20% (w / w) based on the total feed, thereby causing the silkworms to suffer from growth deficiency due to abnormal sugar metabolism; (ii) administering a test substance to the silkworm; (iii) measuring the body weight of the silkworm administered with the test substance; and (iv) selecting a candidate substance capable of treating a glucose metabolism disorder from the test substances based on the body weight of the silkworms; A method comprising:

3. The method according to claim 1 or 2, comprising (i) feeding the silkworms with feed containing sucrose at a concentration of 11% (w / w) or more and 20% (w / w) or less of the total feed for 30 hours or more.

4. (ii) The method according to claim 1 or 2, wherein the test substance is administered orally.

5. 3. The method of claim 1 or 2, comprising (i) and (ii) occurring simultaneously.

6. The method according to claim 1 or 2, wherein the state of growth deficiency due to abnormal glucose metabolism is characterized by one or more of the following symptoms being exhibited in silkworms compared to when the silkworms are fed feed containing no added sucrose: A reduction in one or more of body weight and body size; an increase in the concentration of one or more selected from glucose and trehalose in the hemolymph; Increased concentration of bombyxin in the hemolymph, Insulin resistance, Decreased expression or activity of AMP-activated protein kinase (AMPK).

7. A method for producing a non-human model animal for glucose metabolism disorders, comprising feeding silkworms feed containing sucrose at a concentration of 11% (w / w) or more and 20% (w / w) or less of the total feed for more than 30 hours.

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