Method for evaluating agent for treating lipid metabolism disorders using silkworms and application of the same

Abstract

To provide a method for evaluating effects on improvement of lipid metabolism and anti-obesity effects, or a method for screening a test substance having effects on improvement of lipid metabolism and anti-obesity effects.SOLUTION: There is provided a method for determining whether a test material is a candidate material for treating lipid metabolism disorders, the method including: (i) causing a silkworm to take glucose and making the silkworm in the state of lipid metabolism disorders; (ii) injecting a test material into the silkworm; and (iii) evaluating the lipid metabolism of the silkworm injected with the test material. There is also provided a method for screening a candidate material for treating lipid metabolism disorders, the method further including (iv) selecting a candidate material which can treat lipid metabolism disorders from test materials on the basis of the evaluated lipid metabolism.

Description

[Technical field]

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

[0002] In today's world, where more than 2.1 billion people are said to be overweight or obese, there is still a high demand for pharmaceuticals and functional foods that improve lipid metabolism. In the midst of this, there is a growing movement to prohibit or abolish animal testing using mammals, and there is an urgent need to develop alternative methods to animal testing.

[0003] The construction 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 lipid 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 lipid metabolic disorders in humans 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 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 1 and Non-Patent Document 1 describe that in silkworms fed a high-glucose diet, the amount of triglyceride in the fat body, as well as the amount of triglyceride and the amount of free fatty acid in the body fluid, are increased. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent Publication No. 2015-210100 [Non-patent literature]

[0006] [Non-Patent Document 1] 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. Summary of the Invention [Problem to be solved by the invention]

[0007] However, no method has been found to date using silkworms to evaluate the lipid metabolism improving or anti-obesity effects of drugs for treating dyslipidemia, or food and beverage ingredients that have the effect of inhibiting fat accumulation or promoting lipolysis, or to screen for drugs or food and beverage ingredients that have lipid metabolism improving or anti-obesity effects. [Means for solving the problem]

[0008] The present inventors conducted extensive research to solve the above problems and found that silkworms bred in a certain manner develop obesity and lipid metabolism disorders similar to those seen in mammals. Furthermore, the present inventors found that administering a drug for treating lipid disorders or a food or beverage ingredient having an effect of inhibiting fat accumulation or promoting fat decomposition to this silkworm model can produce the same effects or actions as seen in mammals, leading to the completion of the present invention.

[0009] That is, the present invention relates to the following. [1] A method for evaluating whether a test substance is a candidate substance for treating dyslipidemia, comprising: (i) Inducing abnormal lipid metabolism in silkworms by feeding them sugar; (ii) administering the test substance to the silkworms; and (iii) Evaluating lipid metabolism in silkworms administered the test substance A method comprising: [2] A method for screening a candidate substance for the treatment of dyslipidemia, comprising: (i) Inducing abnormal lipid metabolism in silkworms by feeding them sugar; (ii) administering the test substance to the silkworms; (iii) evaluating the lipid metabolism of silkworms administered the test substance; and (iv) selecting a candidate substance capable of treating lipid abnormalities from the test substances based on the evaluated lipid metabolism. A method comprising: [3] The method according to [1] or [2], wherein sugar is added to the feed and the concentration of sugar in the total feed is 5 to 20% (w / w). [4] The method according to any one of [1] to [3], wherein the sugar is selected from glucose, maltose, sucrose, cellobiose, trehalose, sorbitol, and starch. [5] The method according to any one of [1] to [4], wherein (i) and (ii) are carried out simultaneously. [6] The method according to any one of [3] to [5], wherein the state of lipid metabolism abnormality is exhibited in silkworms by one or more of the following, compared to when the silkworms are fed a feed containing no added sugar: Increased fat mass per unit of body weight, Increased triglyceride content in fat pads, Increased concentration of triglycerides in the hemolymph, an increase in the concentration of one or more selected from glucose and trehalose in the hemolymph; Increased free fatty acid concentration in hemolymph, Increased expression or activity of fatty acid synthesis enzymes in the fat body, Increased expression or activity of rate-limiting enzymes in fatty acid synthesis in the fat body Decreased expression or activity of AMP-activated protein kinase in the fat body. [7] (iii) The method according to any of [1] to [6], wherein the evaluation of lipid metabolism includes one or more selected from the following: measuring the weight of fat bodies; measuring triglycerides in fat bodies; measuring triglycerides in hemolymph; measuring one or more selected from glucose and trehalose in hemolymph; measuring the amount of free fatty acids in hemolymph; measuring the expression or activity of one or more selected from fatty acid synthesis enzymes, fatty acid synthesis rate-limiting enzymes and AMP-activated protein kinase in fat bodies; measuring the activity or expression of lipoprotein lipase in fat bodies or muscle tissue; and measuring the respiratory quotient. Effect of the Invention

[0010] According to the present invention, an evaluation system for lipid metabolism disorders using an insect (silkworm) with few animal ethics issues can be obtained. This system can be useful for evaluating the lipid metabolism treatment effects, fat accumulation inhibition effects, or fat decomposition promotion effects of drugs and food and beverage ingredients, or for screening drugs and food ingredients having lipid metabolism treatment effects, fat accumulation inhibition effects, or fat decomposition promotion effects. [Brief description of the drawings]

[0011] [Figure 1]Figure 1 shows the results of an investigation into the conditions for creating an obese / dyslipidemia silkworm (ODL silkworm) model. Figure 1A: Schedule for feeding, measurement of food intake and body weight, and collection of hemolymph and fat body. Figure 1B: Food intake. Figure 1C: Body weight gain. Figure 1D: Body weight gain per food intake. Figure 1E: Fat body weight per body weight. Figure 1F: Image of fat body. Figure 1G: Triglyceride content in fat body. Figure 1H: Triglyceride concentration in hemolymph. Figure 1I: Free fatty acid concentration in hemolymph. Figure 1J: Sugar concentration in hemolymph. ND: normal feed (Silkmate PM), GD: glucose-supplemented feed. [Diagram 2] Figure 2 shows the results of verification of the lipid metabolism improving effect of hyperlipidemic drugs and anti-obesity functional ingredients in ODL silkworms. Figure 2A: Schedule of feeding silkworms, administration of test substances, weight measurement, hemolymph collection, and fat body collection. Figures 2B-E: Effect of administration of fenofibrate (FB) or epigallocatechin gallate (EGCG) on lipid metabolism improving effect in ODL silkworms. Figure 2B: Sugar concentration in hemolymph. Figure 2C: Neutral fat concentration in hemolymph. Figure 2D: Fat body weight. Figure 2E: Neutral fat content in fat body. [Diagram 3] Figure 3 shows the results of verification of the effects of FB and EGCG on ODL silkworms at the mechanism of action level. Figure 3A: Schedule of feeding silkworms, administration of test substance, weight measurement, blood lymph collection, and fat body collection. Figure 3B-E: Changes in the activity state of fatty acid synthesis enzyme and rate-limiting enzyme of fatty acid synthesis in fat body of ODL silkworms by administration of FB or EGCG. Figure 3C: FAS (Fatty acid synthase), Figure 3D: Inactive acetyl-CoA carboxylase (p-ACC), Figure 3E: Activated AMPK (p-AMPK). Figure 3F-G: Changes in the activity state of lipoprotein lipase (LPL) in fat body or muscle of ODL silkworms by administration of FB or EGCG. Figure 3F: Fat body, Figure 3G: Muscle. [Figure 4]Figure 4 shows the results of examining the change in fatty acid degradation activity by administration of FB or EGCG to ODL silkworms. Figure 4A: Schedule of feeding silkworms, administration of test substance, measurement of respiratory quotient (RQ), hemolymph collection, and fat body collection. Figure 4B: Change in RQ, Figure 4C: Fat body weight per body weight, Figure 4D: Neutral fat content in fat body, Figure 4E: Neutral fat concentration in hemolymph. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention will be described below. The features of the present invention described below can be combined in any manner. In the following, the term "in the present invention" does not refer to a specific embodiment unless otherwise specified.

[0013] <Method for assessing whether a test substance is a candidate substance for treating dyslipidemia> In one aspect, the present invention relates to a method for evaluating whether a test substance is a candidate substance for the treatment of dyslipidemia (hereinafter, sometimes referred to as the evaluation method of the present invention).

[0014] In the present invention, the term "dyslipidemia" refers to a state in which an abnormality occurs in lipid metabolism, and typically refers to a state in which the concentration of lipids in the blood is higher than a standard value, or a state in which the weight of fat pads or the neutral fat content in fat pads is higher than a standard value. The dyslipidemia is, for example, a dyslipidemia in mammals, and preferably a dyslipidemia in humans.

[0015] 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.

[0016] 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.

[0017] (i) Inducing abnormal lipid metabolism in silkworms The evaluation method of the present invention includes (i) causing the silkworm to ingest sugar, thereby causing the silkworm to have lipid metabolism abnormality. Advantages of using silkworms include that they are suitable for evaluating test substances for humans because their pharmacokinetics and sugar metabolism are similar to those of mammals such as humans, their large body size allows multiple administration routes such as oral administration and injection to be selected, there are few animal ethics issues, and the time required for evaluation is short.

[0018] 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.

[0019] The sugar to be ingested by the silkworm is preferably glucose or a sugar containing glucose. Examples of sugars containing glucose include disaccharides such as maltose, sucrose, cellobiose, trehalose, etc., sugar alcohols such as sorbitol, and polysaccharides such as starch. In one embodiment, the sugar to be ingested by the silkworm is glucose.

[0020] In one embodiment, sugar is added to the feed. The concentration of sugar in the feed can be appropriately set, and may be, for example, 5% (w / w) or more, 6% (w / w) or more, preferably 7% (w / w) or more, more preferably 8% (w / w) or more, even more preferably 8% (w / w) or more, particularly preferably 9% (w / w) or more, and most preferably 10% (w / w) or more, or, for example, 20% (w / w) or less, 19% (w / w) or less, 18% (w / w) or less, 17% (w / w) or less, 16% (w / w) or less, 15% (w / w) or less, 14% (w / w) or less, 13% (w / w) or less, 12% (w / w) or less, or 11% (w / w) or less, based on the entire feed. The above-mentioned concentrations may be arbitrarily combined to represent a predetermined concentration range. Furthermore, the phrase "sugar was added to the entire feed at a predetermined concentration" refers to the concentration of sugar added to the entire feed.

[0021] In the present invention, the addition of sugars such as glucose to the feed is not considered to be a requirement of the invention.

[0022] 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.

[0023] The period during which the silkworms are fed with sugar may be, for example, 24 hours or more, preferably 30 hours or more, more preferably 36 hours or more, even more preferably 42 hours or more, and particularly preferably 48 hours or more or 72 hours or more. This period may be, for example, 120 hours or less, preferably 108 hours or less, more preferably 96 hours or less, and even more preferably 84 hours or less.

[0024] In one aspect, a state of abnormal lipid metabolism is characterized by one or more of the following symptoms being exhibited in silkworms compared to when the silkworms are fed a diet without added sugar: Increased fat mass per unit of body weight, Increased triglyceride content in fat pads, Increased concentration of triglycerides in the hemolymph, an increase in the concentration of one or more selected from glucose and trehalose in the hemolymph; Increased free fatty acid concentration in hemolymph, Increased expression or activity of fatty acid synthase (FAS) in fat bodies, Increased expression or activity of rate-limiting enzymes in fatty acid synthesis in the fat body; Decreased expression or activity of AMP-activated protein kinase (AMPK) in the fat body.

[0025] In silkworms, fat bodies exist as tissues that fill the spaces between organs, and those skilled in the art can appropriately collect the fat bodies. For example, as shown in this example, fat bodies can be collected from silkworms by incising the back of a silkworm in a suspended animation state and collecting the exposed white fat body with tweezers or the like.

[0026] The content of neutral fat in the fat body of a silkworm can be measured by any method known to those skilled in the art, for example, by a staining method using a dye that is incorporated into lipid droplets.

[0027] "Hemolymph" corresponds to the body fluids 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 hemolymph leaked from the base of the abdominal legs of silkworm larvae using a syringe needle.

[0028] The concentration of neutral fats in the hemolymph can be measured by any method known to those skilled in the art, for example, by a technique that uses an enzyme reaction specific to neutral fats. 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 technique that uses an enzyme reaction specific to the sugar of interest. The concentration of free fatty acids in the hemolymph can be measured by any method known to those skilled in the art, for example, by a technique that uses an enzyme method specific to free fatty acids.

[0029] The expression or activity of FAS, fatty acid synthesis rate-limiting enzyme (e.g., acetyl-CoA carboxylase (ACC)) or AMPK in fat body can be measured by any method known to those skilled in the art. For example, ACC can be measured by an immunological method using an antibody specific to phosphorylated ACC (p-ACC). Note that the increase in ACC activity may be, for example, an increase in the expression of p-ACC, which is an inactive form.

[0030] In another aspect, the state of dyslipidemia is one or more of the following manifestations in silkworms: The weight of fat pads per body weight is greater than the standard value. The content of neutral fat in fat pads is greater than the standard value. The concentration of neutral fat in the hemolymph is greater 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 free fatty acid concentration in the hemolymph is greater than the standard value. Each reference value can be selected from those known to those skilled in the art from literature, etc.

[0031] (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.

[0032] The dosage of the test substance to silkworms can be appropriately determined by a person skilled in the art depending on the type of test substance or the purpose of evaluation, and may be calculated, for example, by converting the dosage to mammals into the weight of silkworms. When administering the desired dosage 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 test substance.

[0033] The test substance may be administered to the silkworms (1) during the period when the silkworms are ingesting sugar, (2) after the silkworms are ingesting sugar (e.g., after the silkworms have been ingested sugar for a certain period of time, during a period when the silkworms are not ingesting sugar), or (3) before the silkworms are ingesting sugar. 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 ingesting sugar.

[0034] (iii) Evaluating lipid metabolism in silkworms administered the test substance The evaluation method of the present invention includes (iii) evaluating lipid metabolism of silkworms administered with a test substance. In the evaluation method of the present invention, by evaluating lipid metabolism of silkworms administered with a test substance, it is possible to evaluate whether the test substance may be useful for treating lipid abnormalities.

[0035] In one embodiment, the evaluation of lipid metabolism of silkworms may include one or more selected from the following: Measurement of fat pad weight; Measurement of triglyceride content in fat pads; Measurement of triglyceride concentration in hemolymph; Measurement of one or more selected from glucose and trehalose in hemolymph; Measurement of free fatty acid concentrations in hemolymph; measuring the expression or activity of one or more selected from FAS, a rate-limiting enzyme of fatty acid synthesis, and AMPK in fat pads; Measurement of lipoprotein lipase (LPL) activity or expression in fat body or muscle tissue; Measurement of respiratory quotient.

[0036] Measurement of fat pad weight, measurement of triglyceride content in fat pads, measurement of triglyceride concentration in hemolymph, measurement of one or more selected from glucose and trehalose in hemolymph, measurement of free fatty acid concentration in hemolymph, and measurement of expression or activity of one or more selected from FAS, fatty acid synthesis rate-limiting enzyme and AMPK in fat pads can be performed as described above in section (i).

[0037] LPL activity or expression in fat pads or muscle tissue can be measured by any method known to those of skill in the art, and can be measured using an enzymatic reaction that uses an LPL substrate (e.g., 4-methylumbelliferyl oleate).

[0038] Respiratory quotient (RQ) is the ratio of carbohydrate and fat burning to fatty acid oxidation. It is the ratio of oxygen consumption (VO 2 ) and carbon dioxide generation (VCO2 ) is calculated using the following formula: RQ=VCO 2 / VO 2 The respiratory quotient is 1 when only carbohydrates are burned and 0.71 when only lipids are burned. A respiratory quotient greater than 1 indicates that metabolism is geared toward fat synthesis.

[0039] In the evaluation method of the present invention, it can be evaluated that the test substance may be useful for treating lipid abnormalities by improving the lipid metabolism of silkworms. In one embodiment, the improvement of lipid metabolism may be one or more selected from the following, compared to when the test substance is not administered or when the test substance is administered at a lower dose in the silkworms: Loss of fat pad weight; A decrease in the content of triglycerides in fat bodies; A decrease in the concentration of triglycerides in the hemolymph; Decreased concentration of free fatty acids in the hemolymph; Decreased expression or activity of one or more selected from FAS and acetyl-CoA carboxylase (ACC) in the fat body; Increased expression or activity of AMPK in the fat body; Decreased activity or expression of LPL in the fat body; Increased activity or expression of LPL in muscle tissue Decreased respiratory quotient.

[0040] In another embodiment, the improvement in lipid metabolism may be similar to that obtained when the silkworms are administered any positive control known to be capable of treating lipid abnormalities in one or more selected from the following: Changes in fat pad weight; Changes in triglyceride content in fat pads; Changes in the concentration of triglycerides in the hemolymph; Changes in free fatty acid concentrations in hemolymph; altered expression or activity in fat body of one or more selected from FAS, a rate-limiting enzyme of fatty acid synthesis, and AMPK; changes in lipoprotein lipase (LPL) activity or expression in fat body or muscle tissue; Changes in respiratory quotient.

[0041] In the evaluation method of the present invention, it is possible to evaluate whether a test substance can be useful for treating dyslipidemia based on the same mechanism of action as in mammals.

[0042] <Method of screening candidate substances for treating lipid abnormalities> In one aspect, the present invention relates to a method for screening a candidate substance for treating dyslipidemia (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>.

[0043] (iv) selecting a candidate substance capable of treating dyslipidemia from the test substances based on lipid metabolism; The screening method of the present invention includes (iv) selecting a candidate substance capable of treating dyslipidemia from the test substances based on the lipid metabolism evaluated in (iii). The candidate substance capable of treating dyslipidemia is selected from the test substances evaluated as being useful for treating dyslipidemia by evaluating the lipid metabolism of silkworms administered with the test substance in (iii). 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.

[0044] In the screening method of the present invention, candidate substances for treating lipid abnormalities can be screened based on the same mechanism of action as in mammals. EXAMPLES

[0045] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0046] [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.

[0047] Diet preparation The silkworm model was created by feeding silkworms an artificial diet containing added sugar (high sugar diet). A control group was set up where silkworms of the same age were fed Silkmate PM (Normal Diet; ND) without added sugar. ND was prepared according to the product manual, by mixing water and Silkmate PM in a weight ratio of 1:3, kneading well, and then heating at 105°C for 30 minutes using an autoclave (steaming). The high sugar diet was prepared in the same manner as the ND, with the amount of water added reduced by the weight of the sugar to be mixed, and mixed so that the weight ratio of "sugar + water":Silkmate was 1:3. The evaluation samples were mixed into the cooled high sugar diet after autoclaving.

[0048] Blood and tissue sampling 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 a small amount of leakage of hemolymph, which was then collected into a plastic tube and immediately frozen on dry ice. The hemolymph was stored at -20°C until use.

[0049] Fat bodies were collected by placing silkworms in a container filled with dry ice and putting them into a state of suspended animation. Silkworms were cut open from the back to remove the midgut, and the exposed fat bodies were washed with phosphate buffered saline (PBS) and wiped dry, after which all fat bodies were scraped off with tweezers and weighed. The collected fat bodies were frozen in liquid nitrogen and stored at -80°C. After removing the fat bodies, the third to fifth somites (including muscle, dermis, and epidermis) were collected, immediately frozen in liquid nitrogen, and stored at -80°C.

[0050] Measurement of trehalose and glucose concentrations (blood glucose levels) in hemolymph The trehalose concentration in hemolymph was measured as follows. 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 minutes, and then returned to room temperature. 100 μl of this hemolymph solution was mixed with 100 μl of porcine trehalase solution (10 mU / mL) and incubated overnight at 37°C, after which the amount of glucose produced was measured using a Glucose CII Test Wako (Fujifilm Wako Pure Chemical Corporation, Tokyo). The trehalose concentration in the hemolymph was calculated from a calibration curve prepared by measuring trehalose in the same manner.

[0051] 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 the hemolymph was sampled in a 96-well plate placed on dry ice. After sampling, the plate was removed from the dry ice and 200 μl of coloring solution was added immediately. 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.

[0052] Measurement of hemolymph triglyceride concentration Laboratory assays are used to measure triglyceride concentrations in hemolymph. TM Triglyceride (FUJIFILM Wako Pure Chemical Industries, Ltd., Tokyo) was used. 2 μl of hemolymph was placed in each well of a 96-well plate placed on dry ice, 300 μl of color-developing solution was added, and the plate was incubated at 37°C for 5 minutes, after which the absorbance was measured at wavelengths of 600 nm and 700 nm.

[0053] Measurement of free fatty acid concentration in hemolymph To extract free fatty acids from hemolymph, 1 ml of chloroform / ethanol (2:1) solution was added to 40 μl of hemolymph, then stirred and incubated at 37°C for 30 min. 200 μl of water was added, gently stirred, and left to stand at room temperature overnight. The chloroform layer was dried and dissolved in 2% propanol to prepare a sample. The free fatty acid concentration was measured using Labo Assay NEFA (Fujifilm Wako Pure Chemical Corporation, Tokyo). 80 μl of coloring agent A was added to 4 μL of sample, and incubated at 37°C for 10 min. 160 μl of coloring solution B was added, and the mixture was heated at 37°C for 10 min, after which the absorbance at 550 nm was measured.

[0054] 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 ripase 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. One ml of the lysis buffer was added to 50 mg of frozen fat bodies, which were then homogenized with a Polytron homogenizer (Kinematica; Switzerland) for 30 seconds and then allowed to stand on ice for 30 minutes. The protein concentration of the supernatant obtained by centrifuging the lysate (12,000 rpm, 10 min, 4°C) was measured by the BCA method (BCA Protein Assay Kit; Takara Bio Inc., Shiga). After that, 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 so that the protein concentration of each sample was the same. After that, the samples were heat-treated at 90°C for 5 min and subjected to SDS-PAGE using 10% polyacrylamide gel. The proteins in the gel were transferred to a PVDF membrane and reacted with an antibody against the target protein to be detected (primary antibody) and an HRP-labeled secondary antibody against the primary antibody, and then the proteins were analyzed by SuperSignal TM The bands of the target proteins were detected using Chemiliuminescent HRP Substrates (Thermo Fisher Scientific Logo). Band intensities were quantified using Image J.

[0055] Neutral fat staining of fat bodies Immediately after removal, the fat pads were immersed in 4% formaldehyde and fixed overnight at 4°C. Then, the fat pads were fixed in phosphate buffer (137 mM NaCl, 8.1 mM Na 2 HPO 4 , 2.68 mM KCl, 1.47 mM KH 2 PO4 The sections were washed with 60% 2-Propanol (pH 7.4), left to stand in 60% 2-Propanol for 1 minute, and then stained with Oil red O staining solution (1.8 mg of Oil red O in 1 mL of 60% 2-Propanol) for 20 minutes (room temperature). After washing three times with 60% 2-Propanol, the sections were immersed in 100% 2-Propanol and ultrasonicated for 30 minutes. After centrifugation (10,000×g, 3 minutes), the absorbance of the supernatant was measured at 490 nm. The absorbance per gram of fat body was calculated and used as the neutral fat content in each fat body sample.

[0056] Measurement of lipoprotein lipase (LPL) activity Approximately 100 mg of fat body or muscle (including epidermis and dermis) that had been frozen and stored at -80°C was added to 1 ml of ice-cold heparin solution (0.25 M sucrose, 1 mM EDTA, 3 mM Tris-HCl, 2% BSA, 2 U / ml heparin sodium, pH 7.5), and homogenized using a Polytron homogenizer. After incubation at 37°C for 1 hour, the mixture was centrifuged, and the resulting supernatant was used as the enzyme solution. The protein concentration of the enzyme solution was measured using the BCA method. Phosphate buffer (0.2 M Na) was added to each sample so that the protein concentration was the same. 2 HPO 4 12H 2 O, 0.2 M NaH 2 PO 4 2H 2 The enzyme solution was adjusted to 0.3 mM (pH 7.4) and the LPL activity was measured by a fluorescence method using 0.5 mM 4-methylumbelliferyl oleate solution dissolved in phosphate buffer as a substrate. The reaction was started by mixing 100 μl of the enzyme solution and 100 μl of the substrate solution on a 96-well microplate, and the fluorescence intensity (ex / em = 355 / 460) was measured immediately and every 2 minutes for 30 minutes. The fluorescence intensity at 30 minutes divided by the fluorescence intensity at 0 minutes was regarded as the LPL activity.

[0057] Respiratory quotient measurement Respiratory quotient was measured using a metabolic measurement system for small animals (MK-5000RQ / MS, Muromachi Kikai, Tokyo). Measurements were performed at room temperature of 26°C under a 12-hour light-dark cycle. Silkworms were fed with artificial diet containing 10% glucose for 72 hours, and five silkworms were placed in one sealed cage. They were then fed with normal artificial diet with or without the sample. After three hours, oxygen consumption (VO 2 ) and carbon dioxide generation (VCO 2 ) was recorded every 5 minutes for 24 hours. 2 Per VO 2 It was calculated as:

[0058] [result] Construction of an obese and lipid-deficient silkworm (ODL silkworm) model 1.Consideration of conditions for creating the ODL silkworm model Our previous research has revealed that raising silkworms on an artificial diet with a high sugar concentration increases the weight of their fat bodies (a silkworm tissue that functions like a mammalian fat tissue and liver). We hypothesized that silkworms with increased fat body mass could be used to screen for drugs to treat hyperlipidemia and anti-obesity, and conducted a test to verify their usefulness.

[0059] First, we investigated conditions for increasing the weight of silkworm fat bodies. Silkworms on the first day of the fifth instar were fed a normal diet (ND; Silkmate PM) or a normal diet supplemented with 10% glucose (10GD), and food intake, body weight, and fat body weight were measured every 24 hours up to 72 hours (Fig. 1). In addition, hemolymph and fat bodies were collected at 72 hours and the neutral fat content was measured. The concentrations of glucose and trehalose (storage sugar in silkworms) were also measured in the hemolymph.

[0060] At all sampling times, food intake (Fig. 1B) and body weight gain (Fig. 1C) were decreased in the 10GD group compared to the ND group, but body weight gain per unit of food intake was greater in the 10GD group (Fig. 1D).Fat pad weight per unit of body weight was also higher in the 10GD group (Fig. 1E, F), and the value was greatest at 72 h (Fig. 1E).

[0061] After 72 hours of feeding the silkworms on the 10GD diet, the concentrations of neutral lipids in the fat body (Fig. 1G) and hemolymph (Fig. 1H) and the glucose concentrations in the hemolymph (Fig. 1I) were all significantly increased in the 10GD diet compared to the ND group.

[0062] In the silkworms reared on 10GD for 72 hours, the fat body weight and the triglyceride content in the hemolymph and fat body were significantly increased compared to the ND group, suggesting that the silkworms reared on 10GD for 72 hours were developing a condition similar to obesity and lipid metabolism disorders in mammals. Therefore, we next carried out a test to verify the usefulness of the obese and lipid metabolism disorders silkworms as a model animal.

[0063] 2. Verification of the usefulness of the obese and lipid-deficient silkworm (ODL silkworm) as a model animal 2-1) Verification of the lipid metabolism improving effect of hyperlipidemic drugs and anti-obesity functional ingredients in ODL silkworms The ODL silkworms, which were created by rearing them on a 10GD for 72 hours, were given a diet containing the hyperlipidemia treatment drug fenofibrate (FB) and the functional ingredient epigallocatechin gallate (EGCG), which has the effect of improving hyperlipidemia and reducing body fat, and the effects of these were examined (Figure 2).

[0064] In ODL silkworms (non-drug treated group), hemolymph glucose concentration (Fig. 2B), triglyceride content in hemolymph and fat body, and fat body weight were significantly increased compared to the ND group (Fig. 2C-E). In the FB treated group, hemolymph triglyceride concentration (Fig. 2C) was decreased in a concentration-dependent manner compared to the non-treated group. However, no significant difference was observed in fat body weight (Fig. 2D) or triglyceride content in fat body (Fig. 2E). In the EGCG treated group, hemolymph (Fig. 2C) and triglyceride content in fat body (Fig. 2E) were decreased in a concentration-dependent manner compared to the non-treated group. However, no significant difference was observed in fat body weight (Fig. 2D). Hemolymph glucose concentration showed a tendency to decrease compared to the non-treated group for both compounds.

[0065] These results indicate that the effects of both compounds on the ODL silkworm are similar to those on mammals. Therefore, to further verify the usefulness of the ODL silkworm as a model animal, we examined the effects of FB and EGCG on the ODL silkworm at the mechanism of action level.

[0066] 2-2) Verification of the effects of FB and EGCG on ODL silkworms at the mechanism of action level 2-2-1) Activation state of lipid metabolism enzymes The activation state of lipid synthesis enzymes in the fat body of ODL silkworms fed FB and EGCG was evaluated by Western blotting (Fig. 3). Compared to the ND group, the ODL silkworms showed increased expression of FAS (fatty acid synthase), an enzyme that synthesizes fatty acids (Fig. 3B, C), and decreased expression of the inactive form of acetyl-CoA carboxylase (ACC), a rate-limiting enzyme in fatty acid synthesis (Fig. 3B, D), and the active form of AMPK (p-AMPK), which inhibits the activity of FAS and ACC (Fig. 3B, E).

[0067] In the FB-treated group, a significant decrease in FAS expression was observed compared to the non-treated group (Fig. 3B, C). In addition, in the EGCG-treated group, a significant decrease in FAS (Fig. 3B, C) and a significant increase in p-AMPK (Fig. 3B, E) and p-ACC (Fig. 3B, D) were observed compared to the non-treated group (p<0.01, p<0.05). These effects at the molecular level are similar to those of each compound in mammals.

[0068] 2-2-2) Lipoprotein lipase (LPL) activity In mammals, it is known that FB activates lipoprotein lipase (LPL), an enzyme that breaks down neutral fats into fatty acids and glycerol and mediates the incorporation of fatty acids into tissues. It has also been reported that green tea, a food rich in epigallocatechin gallate, reduces LPL expression in adipose tissue and increases LPL expression in muscle. Therefore, we investigated the effects of administration of FB and EGCG to ODL silkworms on LPL in the silkworm fat body and muscle tissues.

[0069] The LPL activity in the ODL silkworms was not significantly different from that in the ND group in either the fat body (Fig. 3F) or muscle tissue (Fig. 3G). In the FB-treated group, the LPL activity in the muscle tended to increase. In the EGCG-treated group, the LPL activity in the fat body was significantly decreased (p<0.05) and the LPL activity in the muscle was significantly increased (p<0.01) compared to the ND group. These results indicate that both compounds have the same effect on the LPL activity in silkworms as on the LPL activity in mammals.

[0070] 2-2-3) Fatty acid decomposition activity The lipid metabolism disorder improving effect of FB and EGCG is also exerted through the promotion of fatty acid β-oxidation. Therefore, to verify whether the same effect is observed in ODL silkworms, the respiratory quotient (RQ), which is an index of fatty acid degradation activity, was measured. Silkworms that were raised on 10GD for 48 hours to induce obesity and lipid metabolism disorders were fed FB or EGCG mixed with their normal diet from the 48th hour until the 75th hour. During this period, the ODL silkworms' respiratory quotient (RQ) was increased by 10%. 2 Consumption and CO 2 The CO excretion was measured using a metabolic measurement system for small animals. 2 Emissions O 2 Calculated by dividing by the consumption (RQ=VCO 2 / VO 2 After switching to the normal diet, data were not collected for 3 hours to allow the silkworms to acclimate to the measurement environment, and data were collected for 24 hours from 51 to 75 hours (Figure 4A).

[0071] RQ was divided into three 24-hour periods and the average value for each 8-hour period was shown (Fig. 4B). RQ is an index that estimates the amount of nutrients being burned, and is 1 when only carbohydrates are burned and 0.71 when only lipids are burned. A value above 1 indicates that metabolism is moving toward fat synthesis. The RQ in ODL silkworms decreased over time from the start of the measurement, but in the non-administered group, it exceeded 1 even at 17-24 hours after the start of the measurement, suggesting that fat synthesis exceeded nutrient breakdown. On the other hand, in the FB and EGCG-administered groups, the RQ for 17-24 hours was below 1, suggesting that fat breakdown exceeded fat synthesis. The RQ for 17-24 hours was the lowest in the EGCG group, which was consistent with the fact that the EGCG group had the largest decrease in hemolymph and fat body triglyceride content among the three groups (Fig. 4D, E). However, no significant difference was observed in fat body weight (Fig. 4C).

[0072] The results so far have shown that the lipid-lowering drug fenofibrate and the functional ingredient epigallocatechin gallate, which has lipid-lowering and lipolytic effects, act on the obese and lipid-depleted silkworm (ODL silkworm) through the same mechanism as in mammals. The ODL silkworm has been shown to be a useful model animal for screening materials with lipid-lowering effects. [Industrial Applicability]

[0073] The evaluation method and screening method of the present invention using silkworms may be a useful alternative to animal testing for evaluating lipid metabolism as animal testing is increasingly phased out.

Claims

1. 1. A method for evaluating whether a test substance is a candidate substance for the treatment of dyslipidemia, comprising: (i) causing the silkworm to have abnormal lipid metabolism by feeding the silkworm with sugar; (ii) administering a test substance to the silkworm; and (iii) Evaluating lipid metabolism in silkworms administered the test substance A method comprising:

2. 1. A method for screening a candidate substance for the treatment of dyslipidemia, comprising: (i) causing the silkworm to have abnormal lipid metabolism by feeding the silkworm with sugar; (ii) administering a test substance to the silkworm; (iii) Evaluating lipid metabolism of silkworms administered the test substance; and (iv) selecting a candidate substance capable of treating dyslipidemia from the test substances based on the evaluated lipid metabolism; A method comprising:

3. The method according to claim 1 or 2, wherein sugar is added to the feed, and the concentration of sugar in the total feed is 5 to 20% (w / w).

4. 3. The method of claim 1 or 2, wherein the sugar is selected from glucose, maltose, sucrose, cellobiose, trehalose, sorbitol, and starch.

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

6. The method according to claim 3, wherein the state of lipid metabolism abnormality is characterized by one or more of the following symptoms being exhibited in silkworms compared to when the silkworms are fed a feed containing no added sugar: Increased fat mass per unit of body weight, Increased triglyceride content in fat pads, Increased concentration of triglycerides in the hemolymph, an increase in the concentration of one or more selected from glucose and trehalose in the hemolymph; Increased free fatty acid concentration in hemolymph, Increased expression or activity of fatty acid synthesis enzymes in the fat body, Increased expression or activity of rate-limiting enzymes in fatty acid synthesis in the fat body Decreased expression or activity of AMP-activated protein kinase in the fat body.

7. (iii) The method of claim 1 or 2, wherein the evaluation of lipid metabolism includes one or more selected from the following: measuring the weight of the fat body; measuring the triglyceride content in the fat body; measuring the triglyceride concentration in the hemolymph; measuring the concentration of one or more selected from glucose and trehalose in the hemolymph; measuring the concentration of free fatty acids in the hemolymph; measuring the expression or activity of one or more selected from fatty acid synthesis enzyme, fatty acid synthesis rate-limiting enzyme and AMP-activated protein kinase in the fat body; measuring the activity or expression of lipoprotein lipase in the fat body or muscle tissue; and measuring the respiratory quotient.

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