JP6388786B2 - Evaluation method, screening method, and production method of prophylactic / therapeutic agent for type 2 diabetes using hyperglycemic silkworm - Google Patents

Description

  The present invention relates to a method for evaluating, screening and producing a prophylactic / therapeutic agent for type II diabetes using “hyperglycemic silkworm”.

Diabetes mellitus is a disease that shows a chronic hyperglycemic state and abnormal glucose tolerance, and causes various organ disorders such as nephropathy, peripheral neuropathy, and retinopathy (Non-patent Document 1). Insulin, which is a peptide hormone, has a remarkable hypoglycemic effect and has been used as a therapeutic drug for diabetes.
In recent years, however, the number of patients with type II diabetes caused by insulin resistance has been increasing in developed countries. In mammals such as humans, rats, and mice, it is said that they become obese by eating a high-calorie diet for a long period of time, and type II diabetes develops (Non-patent Document 1). Drugs that lower blood sugar levels by improving insulin resistance have been used for the treatment of type II diabetes, but have the problem of causing drug resistance and side effects, and the development of new therapeutic drugs is desired.

  Blood glucose levels are maintained by sugar uptake and metabolism in systemic tissues. Therefore, in order to evaluate the effects of antidiabetic drugs, it is necessary to construct a disease state model using animals that can quantitatively measure blood glucose levels. 2. Description of the Related Art Conventionally, diabetes therapeutic drugs using diabetes pathological models by mammals such as mice and rats have been developed. However, sacrificing a large number of mammals has been pointed out to be problematic from the viewpoint of animal welfare as well as high rearing costs, and has become a limiting factor for therapeutic drug development.

It is known that invertebrates such as insects also have insulin transmission pathways common to mammals, and blood glucose levels are regulated. In silkworms, it is believed that a peptide hormone called insulin, which is homologous to insulin, lowers blood glucose levels.
The present inventors have reported that the hypoglycemic action of human insulin, which is a therapeutic agent for diabetes, can be evaluated using silkworms that have been hyperglycemic by giving a high glucose diet for a short time (Patent Documents 1, 2, Non-patent document 2). That is, using hyperglycemic silkworms, it is possible to evaluate compounds such as human insulin that have an immediate hypoglycemic effect. Silkworms have the advantage that they can be raised in large quantities in a small, inexpensive space with few problems of animal welfare.

In addition, the present inventors have been able to evaluate a therapeutic agent for bacterial infections and a therapeutic agent for viral infections using a silkworm infection model, and have aspects common to mammals and silkworms in the disposition of antibiotics. It has been reported (Patent Document 3, Non-Patent Document 3). Silkworms are characterized by the ability to easily perform quantitative intra-blood injection of drugs using a syringe, which is difficult for small invertebrates such as nematodes and fruit flies. It is also easy to collect blood from silkworms and analyze biochemical properties in body fluids such as measuring blood sugar levels.
Furthermore, the present inventors have succeeded in identifying a hypoglycemic substance that has an effect on a diabetic mouse from a herbal medicine, taking advantage of a hyperglycemic silkworm that can be easily experimented with a large number of individuals ( Patent Documents 1 and 2, Non-Patent Document 2).

  On the other hand, even when pioglitazone, a therapeutic agent for type II diabetes, was administered to silkworms that became hyperglycemic by giving a high glucose diet for a short time, no immediate decrease in blood glucose level was observed. This is because the hyperglycemia model of silkworms by feeding a high glucose diet for a short time decreases the blood glucose level by insulin administration, and the blood glucose level decreases several hours after the start of fasting after administration of the high glucose diet. It is thought that this is caused by not simulating the type II diabetes model.

  In recent years, there has been an increasing demand for preventive or therapeutic agents for human type II diabetes, and conventional evaluation methods, screening methods, and production methods using them are not sufficient, and further improvements have been made. It was desired.

JP 2009-058500 A International Publication No. 2010/004916 JP 2010-133975 A

Catherine Carver, The Diabetes EDUCATOR, 2006, vol. 32, 910-916 Matsumoto Y, Sumiya E, Sugita T, Sekimizu K. An Invertebrate Hyperglycemic Model for the Identification of Anti-Diabetic Drugs. PLoS ONE, 6 (3), 2011 Matsumoto Y, Miyazaki S, Fukunaga DH, Shimizu K, Kawamoto S, Sekimizu K. Quantitative evaluation of cryptococcal pathogenesis and antifungal drugs using a silkworm infection model with Cryptococcus neoformans. J Appl Microbiol. 112 (1): 138-46 (2012)

The present invention has been made in view of the above-mentioned background art, and its task is to construct a model animal with few ethical problems for evaluating a therapeutic agent for type II diabetes, and the test substance is human type II diabetes It is an object of the present invention to provide a method for evaluating whether a substance is a candidate substance for preventing or treating.
Another object of the present invention is to provide a method for screening a substance for preventing or treating human type II diabetes using the model animal, and a method for producing a drug for preventing or treating human type II diabetes.

  As a result of intensive studies to solve the above-mentioned problems, the present inventor has developed (i) an abnormality of blood sugar during fasting, It was found for the first time that the symptoms of human type II diabetic patients can be exhibited, such as ii) decreased glucose tolerance at the individual level, (iii) abnormal lipid metabolism, and (iv) increased fat body insulin resistance. .

  The present inventors have also found that fasting blood glucose levels are reduced when pioglitazone or metformin, which is a therapeutic agent for type II diabetes, is administered to the silkworm. That is, for the first time using an invertebrate, the present inventors have found that it is possible to evaluate the hypoglycemic effect of a therapeutic agent for type II diabetes.

That is, the present invention is a method for evaluating whether a test substance is a candidate substance for preventing or treating human type II diabetes,
(A) by allowing the silkworm to ingest sugar (A), to at least one state selected from the group consisting of fasting blood glucose abnormality, glucose tolerance abnormality, insulin resistance, and lipid abnormality;
(B) In the silkworm obtained in the step (a), at least one silkworm selected from the group consisting of abnormal fasting blood glucose, abnormal glucose tolerance, insulin resistance, and lipid abnormality, Administering a test substance;
(C) fasting a silkworm administered with the test substance,
(D) a step of measuring the concentration of sugar (B) in the fat body or blood of the fasted silkworm,
To provide a method characterized by comprising:

Further, the present invention is a method for screening a substance for preventing or treating human type II diabetes,
(A) by allowing the silkworm to ingest sugar (A), to at least one state selected from the group consisting of fasting blood glucose abnormality, glucose tolerance abnormality, insulin resistance, and lipid abnormality;
(B) In the silkworm obtained in the step (a), at least one silkworm selected from the group consisting of abnormal fasting blood glucose, abnormal glucose tolerance, insulin resistance, and lipid abnormality, Administering a test substance;
(C) fasting a silkworm administered with the test substance,
(D) a step of measuring the concentration of sugar (B) in the fat body or blood of the fasted silkworm,
(E) selecting a substance that reduces the concentration of sugar (B) in the fat body or blood of the silkworm from the test substance,
To provide a method characterized by comprising:

Further, the present invention is a method for producing a prophylactic or therapeutic agent for human type II diabetes,
(A) by allowing the silkworm to ingest sugar (A), to at least one state selected from the group consisting of fasting blood glucose abnormality, glucose tolerance abnormality, insulin resistance, and lipid abnormality;
(B) In the silkworm obtained in the step (a), at least one silkworm selected from the group consisting of abnormal fasting blood glucose, abnormal glucose tolerance, insulin resistance, and lipid abnormality, Administering a test substance;
(C) fasting a silkworm administered with the test substance,
(D) a step of measuring the concentration of sugar (B) in the fat body or blood of the fasted silkworm,
(E) selecting a substance that reduces the concentration of sugar (B) in the fat body or blood of the silkworm from the test substance,
(F) mixing the substance selected in step (e) with a pharmaceutically acceptable carrier;
To provide a method characterized by comprising:

  According to the present invention, silkworms that solve the above-mentioned problems and the above-mentioned problems, are advantageous in terms of cost, have few ethical problems, and are easy to breed are used. The “examination / evaluation on the candidate substances to be shown” and “various pharmacological experiments” can be easily, inexpensively, efficiently, accurately and appropriately.

  About the "evaluation method and screening method of a hypoglycemic substance in mammals" using a silkworm that has become hyperglycemic by giving a high glucose diet, the present inventors have already reported (Patent Document 1). However, as a result of extensive studies after filing for such a “hyperglycemic silkworm”, no immediate decrease in blood glucose level was observed even when pioglitazone, a type II diabetes treatment, was administered. . In other words, in silkworms used in the previous evaluation methods, mammals improved fasting blood glucose abnormalities, suppressed glucose tolerance, improved insulin resistance, type II diabetes prevention / treatment, chronic glucose metabolism disorders Whether or not it becomes a candidate substance for prevention / treatment can not be correctly evaluated, and it has not been considered to be used for evaluation of at least prevention / treatment of the above-mentioned symptoms.

However, for the first time by the evaluation method of the present invention, it was confirmed that the blood glucose level decreased with both pioglitazone and metformin, which are known as human type II diabetes therapeutic agents. Therefore, for the first time according to the present invention, a silkworm can be used to screen a substance for preventing or treating human type II diabetes.
In addition, the silkworm used in the present invention is confirmed to be in a state of abnormal blood glucose, impaired glucose tolerance, insulin resistance, and lipid abnormality in the fasting state, that is, that human type II diabetes-like symptoms appear. I have confirmed. Silkworms that have been used conventionally do not simulate a human type II diabetes model because the blood glucose level decreases due to insulin administration and the blood glucose level decreases several hours after the start of fasting after administration of high glucose diet.
That is, unlike the silkworm used in the conventional evaluation method and the silkworm used in the evaluation method of the present invention, the evaluation method of the present invention is novel and advanced compared to the conventional evaluation method and the like. Have sex.

Moreover, it is said that it is necessary to give a high fat diet for 2 weeks or more in order to show type II diabetes-like symptoms in mice. On the other hand, a type II diabetes model silkworm can be constructed in a simpler and shorter period than a type II diabetes model mouse.
In addition, nematodes and Drosophila used as pathological model animals are difficult to measure blood glucose because blood sampling is not easy. On the other hand, silkworms are suitable for type II diabetes model animals because blood sampling and blood sugar level measurement are easy.

(A) It is the figure which showed the scheme for constructing a type II diabetes model silkworm. (B) It is a graph which shows the blood glucose level after giving the food containing 10% (w / w) glucose for 18 hours. (C) It is a graph which shows the blood glucose level of the silkworm which fasted for 24 hours, after feeding the food containing 10% (w / w) glucose for 18 hours. “ND” indicates a normal feed group, and “GD” indicates a high glucose feed group. The vertical axis represents the blood sugar concentration (Hemolymph sugar). (A) It is the figure which showed the experimental scheme for verifying the fall of the glucose tolerance of the silkworm which gave glucose feed for a fixed period and was made into hyperglycemia. (B, C) It is the graph which showed the result of having performed the glucose tolerance test to the silkworm which fasted for 24 hours, after feeding the food containing 10% (w / w) glucose for 18 hours. (B) is a graph showing changes in the total sugar concentration in the blood, and (C) is a graph showing changes in the glucose concentration in the blood (Hemolymph glucose). It is the figure which showed the result of having verified whether the phosphorylation of Akt by insulin was induced | guided | derived in the fat body of the silkworm which gave glucose feed for a certain period and was made hyperglycemia. “P-Akt” shows the results of Western blotting using “anti-phosphorylated Akt antibody”, and “Total Akt” shows the results of Western blotting using “anti-Akt antibody”. (A) It is the figure which showed the result which carried out Oil Red O dyeing | staining of the fat body of the silkworm which gave glucose feed for a fixed period and was made hyperglycemia. (B) It is a graph which shows the fixed_quantity | quantitative_assay result of the pigment | dye extracted by processing the fat body dye | stained by said (A) with xylene. The amount of triglycerides in fat bodies (A), the amount of free fatty acids in fat bodies (B), the amount of triglycerides in body fluids (C), the amount of free fatty acids in body fluids It is the graph which showed the result of having quantified (D). It is the figure which showed the result of having verified whether the phosphorylation of JNK was accelerated | stimulated in the fat body of the silkworm which gave glucose feed for a fixed period and was made into the hyperglycemia. “P-JNK” shows the results of Western blotting using “anti-JNK phosphorylated antibody”, and “Total JNK” shows the results of Western blotting using “anti-JNK antibody”. (A) A diagram showing an experimental scheme for verifying whether pioglitazone or metformin used as a therapeutic agent for type II diabetes lowers blood glucose level in silkworms that have been hyperglycemic by giving glucose feed for a certain period of time. is there. (B, C) After administration of pioglitazone (B) or metformin (C), it is the figure which showed the measurement result of the blood glucose level in the fasting. It is a graph which shows the measurement result of the blood glucose level 6 hours after administering and administering to pioglitazone (A) or metformin (B) to the silkworm which gave glucose feed for a short time and made it hyperglycemia.

  Hereinafter, the present invention will be described, but the present invention is not limited to the following specific embodiments, and can be arbitrarily modified within the scope of the technical idea.

[Method of evaluating candidate substances for preventing or treating type II diabetes using a type II diabetes model silkworm]
The evaluation method of the present invention is a method for evaluating whether or not a test substance is a candidate substance for preventing or treating human type II diabetes,
(A) by allowing the silkworm to ingest sugar (A), to at least one state selected from the group consisting of fasting blood glucose abnormality, glucose tolerance abnormality, insulin resistance, and lipid abnormality;
(B) In the silkworm obtained in the step (a), at least one silkworm selected from the group consisting of abnormal fasting blood glucose, abnormal glucose tolerance, insulin resistance, and lipid abnormality, Administering a test substance;
(C) fasting a silkworm administered with the test substance,
(D) a step of measuring the concentration of sugar (B) in the fat body or blood of the fasted silkworm,
It is the method characterized by having.

  The evaluation method may further include other steps as necessary. Hereinafter, steps (a), (b), (c), and (d) will be described in order.

<Process (a)>
In the step (a), first, the silkworm is ingested with the sugar (A), and the silkworm is at least one selected from the group consisting of fasting blood glucose abnormality, glucose tolerance abnormality, insulin resistance, and lipid abnormality. To the state.

The silkworm used in the present invention has the following advantages.
(1) It is easy to obtain silkworms themselves.
(2) A method for rearing silkworms has already been established, and it is convenient for rearing.
(3) Properties similar to the internal organs and organs of mammals such as humans have been known to some extent in previous studies.
(4) Genetic lines have been established and genetic homogeneity has been maintained.
(5) Since it is relatively large, slowly moving, and substantially hairless, it is easy to administer drugs, such as being able to inject quantitatively.
(6) Having fat bodies, the fat bodies can be taken out and the contained substances can be quantified.
(7) It is cheaper than mice, rats, etc., and many individuals can be bred in a small space, and there are few ethical problems, so it is easy to perform screening evaluation.
(8) Evaluation can be performed even when there is only a small amount of the test substance.
(9) It is easy to align individuals in the same state, such as aligning ages.
(10) It is possible to collect body fluids and analyze components such as sugars, lipids and enzymes.

  The silkworm is preferably a large silkworm from the viewpoint of ease of intake of sugar (A), ease of administration of the test substance, ease of collection of blood (body fluid) and fat body, and the like. Here, the “large silkworm” is a silkworm having a body length of 1 cm or more, preferably 1.5 cm or more and 15 cm or less, particularly preferably 2 cm or more and 5 cm or less. Further, silkworms of 4th to 5th instars are preferable, and 5th instar larvae are particularly preferable.

  There is no restriction | limiting in particular as a method of ingesting sugar (A), According to the objective, it can select suitably. Specifically, for example, injection into blood, oral ingestion by addition to feed (food), injection into the intestine, etc. are mentioned, and in terms of simplicity and correspondence with human clinical practice. Oral ingestion by injection into the intestinal tract, addition to feed (food) or the like is preferable.

  As sugar (A), by ingesting sugar (A), silkworm has at least one state selected from the group consisting of fasting blood glucose abnormality, glucose tolerance abnormality, insulin resistance, and lipid abnormality. As long as it is caused, it is not necessary to be the same as sugar (B) and is not particularly limited. For example, monosaccharides such as glucose, heptose, hexose, pentose, tetrose, triose; trehalose, maltose, lactose, sucrose Disaccharides such as cellobiose, nigerose and sophorose; oligosaccharides such as fructooligosaccharides, galacto-oligosaccharides and dairy oligosaccharides; polysaccharides such as starch, glycogen, cellulose, pectin and glucomannan; deoxy sugars; sugars such as uronic acid Oxidation products; sugar alcohols such as sorbit and arabit Among these, when glucose, sucrose or starch is ingested by silkworm, at least one state selected from the group consisting of abnormal fasting blood glucose, abnormal glucose tolerance, insulin resistance, and lipid abnormality in the silkworm And is particularly preferable in terms of correspondence with human clinical practice, and more preferably glucose. These may be used alone or in combination of two or more.

  There is no restriction | limiting in particular as an ingestion amount, According to the substance to ingest, an ingestion method, etc., it can select suitably. In the present invention, when the substance to be ingested is added to the feed (feed), the content ratio of sugar (A) is 2 mass of sugar (A) with respect to the total amount of feed (feed) and sugar (A). % To 30% by mass is preferable, 5% to 25% by mass is more preferable, and 8% to 12% by mass is particularly preferable. If the content is too small, it may not be possible to cause fasting blood glucose abnormalities, glucose tolerance abnormalities, insulin resistance, or lipid abnormalities. May cause disability.

  The ingestion period is not particularly limited as long as the silkworm can be brought into at least one state selected from the group consisting of abnormal fasting blood glucose, abnormal glucose tolerance, insulin resistance, and lipid abnormality. Time to 5 days are preferable, 10 hours to 3 days are more preferable, and 15 hours to 24 hours are particularly preferable. If the intake time is too short, the silkworm may not be in at least one of the above states, whereas if it is too long, the silkworm may grow into cocoons.

  As the feed (food), mulberry leaves, artificial feed (Silk Mate 2S, manufactured by Nippon Agricultural Industry Co., Ltd.) and the like are preferable. Among these, when artificial feed is used, sugar and sugar (A) contained in the artificial feed It is preferable to mix and contain sugar (A) in the ratio of 5 mass%-25 mass% with respect to the total amount of, and it is especially preferable to make it contain in the ratio of 8 mass%-20 mass%. The intake period is preferably 10 hours to 5 days, particularly preferably 15 hours to 24 hours.

  In the step (a), it is essential to set the silkworm to at least one state selected from the group consisting of fasting blood glucose abnormality, glucose tolerance abnormality, insulin resistance, and lipid abnormality.

  Type II diabetes is a disease that results from impaired insulin action (insulin resistance) in target cells, including fasting blood glucose abnormalities, impaired glucose tolerance, lipid metabolism abnormalities (lipid abnormalities), increased oxidative stress (reactive oxygen levels) Increase), a decrease in liver function, and a decrease in kidney function.

Human fasting blood glucose abnormality refers to a condition in which there is an abnormality in the value of fasting blood glucose. It is a test index for glucose metabolism function and is one of diagnostic indexes for diabetes and the like.
The abnormal glucose tolerance in humans means that glucose loaded in the blood is normally removed from the blood within a certain period, but the blood glucose concentration remains high for a certain period. Whether or not the glucose tolerance is abnormal can be confirmed by a human glucose tolerance test or the like.
Human insulin resistance refers to the fact that blood glucose concentration does not decrease despite insulin being secreted or administered.
Human lipid abnormality refers to a condition in which the value of lipids (cholesterol, triglycerides, free fatty acids, etc.) in blood is abnormal.

  From the results shown in the Examples, and the like, in mammals as well as mammals, eating a high glucose diet increased the amount of triglycerides in the fat body and accordingly increased the amount of free fatty acids in body fluids. Conceivable. It is thought that the free fatty acid induced phosphorylation of Akt in fat body cells, causing insulin resistance, and ultimately causing a decrease in glucose tolerance of the fat body.

  In the present invention, “the state of abnormal fasting blood glucose in silkworms” is as described in the examples. That is, it means a state in which the blood sugar level of a silkworm fasted for a certain period of time is higher than that of a normal silkworm.

In the present invention, “the state of abnormal glucose tolerance in silkworms” is as described in the examples. That is, it refers to a state in which the total sugar concentration or glucose concentration in blood maintains a high value for a certain period by administering (loading) glucose into the blood of silkworms.
In addition, the “insulin resistance state of silkworm” is as described in the examples. That is, Akt in the silkworm fat body is a state in which phosphorylation is not induced even when insulin is administered.
Further, “the state of abnormal lipid in silkworm” is as described in the examples. That is, it means a state in which at least one of the triglyceride concentration in the fat body of silkworm, the free fatty acid concentration in the body fluid, and the triglyceride concentration in the body fluid is increased compared to the normal time.

  Silkworms used in the present invention are easy to handle quantitatively compared to mammals such as mice. That is, in order to cause a type II diabetes-like symptom to mammals such as mice, it is necessary to give a high fat diet for 2 weeks or more. On the other hand, it was found from the examples of the present application that a type II diabetes model silkworm can be easily produced. Also in this respect, invertebrates such as silkworms are superior as evaluation animals compared to mammals such as mice.

<Step (b)>
In the step (b), at least one silkworm in the state selected from the group consisting of “fasting blood glucose abnormality, glucose tolerance abnormality, insulin resistance, and lipid abnormality” obtained in the step (a) is obtained. The test substance is administered.

  There is no restriction | limiting in particular as a method of administering a test substance, According to the objective, it can select suitably. Specifically, for example, injection into blood, oral intake by addition to feed (food), injection into the intestine, and the like can be mentioned. In terms of simplicity and compatibility with human clinical practice, injection into the intestinal tract and oral ingestion are preferred.

  There is no restriction | limiting in particular as a dose of a to-be-tested substance, According to the substance to administer, administration method, etc., it can select suitably. It is also preferable to administer the dose per human body weight in terms of the weight of the silkworm to be administered. It is also preferable to administer an amount obtained by multiplying the converted value by a predetermined magnification. It is also preferable to administer the test substance diluted with physiological saline, water or the like.

  The administration time of the test substance is not particularly limited. Immediately after the intake of sugar (A) in step (a), in the silkworm, fasting blood glucose abnormality, glucose tolerance abnormality, insulin resistance, and lipid abnormality At least one state selected from the group, and the silkworm is selected from the group consisting of fasting blood glucose abnormality, glucose tolerance abnormality, insulin resistance, and lipid abnormality The point in time when at least one state is reached is preferable in terms of obtaining reproducibility of the experimental results. Specifically, for example, a period between “immediately after completing the intake of sugar (A)” and “after 24 hours from the completion of intake of sugar (A)” is preferable, and “after completing the intake of sugar (A)” The period from “immediately after the intake of sugar (A) to 6 hours after the completion of intake of sugar (A)” is more preferable, and “immediately after the completion of intake of sugar (A)” is particularly preferable.

  The administration period is not particularly limited as long as the decrease in “concentration of sugar (B) in fat body or blood” by the test substance can be measured, but it is convenient to administer the whole dose at once or continuous administration. This is preferable.

<Step (c)>
In the step (c), the silkworm administered with the test substance in the step (b) is fasted. Specifically, removal of food from the breeding container, movement of the silkworm to another breeding container in which no food is present, and the like can be mentioned.

  The period for fasting is not particularly limited, and may be selected from the period immediately after administration of the test substance in step (b) until the digestion of sugar (A) is completed. Specifically, it is sufficient if the fasting blood glucose level can be measured. For example, 3 hours to 4 days are preferable from the time when the test substance is administered, 10 hours to 3 days are more preferable, and 12 hours to 2 days are preferable. If the fasting period is too short, sugar (A) digestion may not be completed, while if it is too long, it may cause a disorder other than type II diabetes-like symptoms.

<Step (d)>
In the step (d), the concentration of the sugar (B) in the fat body or blood of the silkworm fasted in the step (c) is measured. As for the method of collecting the measurement sample, it is preferable to dissect and collect the fat body, and for blood, the method of collecting from a cut is preferable.

  As a method for measuring the concentration of sugar (B), there is no particular limitation on the method for determining sugar. For example, the phenol sulfate method, the anthrone sulfate method, the carbazole sulfate method, etc. are used for the determination of all sugars; Examples include glucose oxidase method. There is no particular limitation on the time when the concentration of sugar (B) in the fat body or blood is measured, and immediately after the test substance is administered in step (b) (immediately after the start of fasting in step (c)). From the period until the effect of decreasing the concentration of sugar (B) by the test substance is no longer observed. Specifically, for example, 3 hours to 4 days from the time of administration of the test substance is preferable, 10 hours to 3 days are more preferable, and 12 hours to 2 days are particularly preferable.

  In measuring the concentration of sugar (B), at least one or more selected from the group consisting of “fasting blood glucose abnormality, glucose tolerance abnormality, insulin resistance, and lipid abnormality” is not administered. It is preferable to use “infested silkworm” as a control. For the control, for example, it is preferable to administer only the same amount of physiological saline. The test substance is evaluated according to how much the concentration of the sugar (B) is decreased in the test substance administered as compared with the control.

  When pioglitazone and metformin were used as the test substances, a decrease in the sugar (B) concentration was observed. The method for evaluating a candidate substance for preventing or treating type II diabetes using the type II diabetes model silkworm of the present invention has an effect of preventing or treating human type II diabetes on a certain substance without any cost or ethical problem. It is a method that can evaluate whether or not there is.

  Candidate substances for preventing or treating human type II diabetes include candidate substances for improving fasting blood glucose abnormalities in humans, candidate substances for suppressing impaired glucose tolerance, candidate substances for improving insulin resistance, chronic glucose metabolism It is also a candidate substance for preventing and treating disorders.

[Screening method for substances that prevent or treat type II diabetes using type II diabetes model silkworm]
The screening method of the present invention is a method for screening a substance for preventing or treating human type II diabetes,
(A) by allowing the silkworm to ingest sugar (A), to at least one state selected from the group consisting of fasting blood glucose abnormality, glucose tolerance abnormality, insulin resistance, and lipid abnormality;
(B) In the silkworm obtained in the step (a), at least one silkworm selected from the group consisting of abnormal fasting blood glucose, abnormal glucose tolerance, insulin resistance, and lipid abnormality, Administering a test substance;
(C) fasting a silkworm administered with the test substance,
(D) a step of measuring the concentration of sugar (B) in the fat body or blood of the fasted silkworm,
(E) selecting a substance that reduces the concentration of sugar (B) in the fat body or blood of the silkworm from the test substance,
It is the method characterized by having.

  The screening method of the present invention may further include other steps as necessary. Steps (a), (b), (c) and (d) are the same as in the evaluation method.

<Process (e)>
In the step (e), the concentration of the sugar (B) in the fat body or blood of the silkworm from the test substances used in the steps (a), (b), (c) and (d). Select a substance that lowers. Compared to the control, the test substance was administered. When the sugar (B) concentration was reduced to what extent, the test substance was selected as a significant difference. Although it depends on the number and is not particularly limited, it decreased to 70% with bioglitazone and decreased to 80% with metformin (Example 5).

  The number of silkworms used in one condition is not particularly limited, but is preferably 1 to 200, and particularly preferably 2 to 10. Within this range, pharmacologically and statistically correct selection is possible.

  According to the screening method of the present invention, it is possible to screen for “substances for preventing or treating human type II diabetes” that do not depend on a decrease in insulin secretion, in a cost-effective manner and with no ethical problem. That is, it is possible to screen for substances that prevent or treat fasting blood glucose abnormalities, glucose tolerance abnormalities, insulin resistance, or lipid abnormalities that are pathological conditions of type II diabetes.

  Substances that prevent or treat human type II diabetes include substances that improve fasting blood glucose abnormalities, substances that suppress impaired glucose tolerance, substances that improve insulin resistance, and prevent chronic glucose metabolism disorders・ It becomes a substance to treat.

[Method for producing a drug for preventing or treating human type II diabetes using a type II diabetes model silkworm]
The method for producing a drug for lowering blood glucose level according to the present invention is a method for producing a prophylactic or therapeutic agent for human type II diabetes,
(A) by allowing the silkworm to ingest sugar (A), to at least one state selected from the group consisting of fasting blood glucose abnormality, glucose tolerance abnormality, insulin resistance, and lipid abnormality;
(B) In the silkworm obtained in the step (a), at least one silkworm selected from the group consisting of abnormal fasting blood glucose, abnormal glucose tolerance, insulin resistance, and lipid abnormality, Administering a test substance;
(C) fasting a silkworm administered with the test substance,
(D) a step of measuring the concentration of sugar (B) in the fat body or blood of the fasted silkworm,
(E) selecting a substance that reduces the concentration of sugar (B) in the fat body or blood of the silkworm from the test substance,
(F) mixing the substance selected in step (e) with a pharmaceutically acceptable carrier;
It is the method characterized by having.

  The method for producing a drug for lowering blood glucose level according to the present invention may further include other steps as necessary. Steps (a), (b), (c), (d) and (e) are the same as in the above screening method.

<Step (f)>
Step (f) is a step of mixing the substance selected in step (e) and a pharmaceutically acceptable carrier.

  The “substance for preventing or treating human type II diabetes” found by the evaluation method and the screening method of the present invention is used to produce a drug. There are no particular limitations on the dosage form of the drug, but preparations for oral administration include tablets, pills, granules, capsules, powders, solutions, suspensions, syrups, sublinguals, etc. Examples of the preparation for parenteral administration include injections, transdermal absorption agents, inhalants, suppositories and the like.

  In formulating, a pharmaceutically acceptable carrier can be mixed. The type and composition of the carrier can be appropriately determined depending on the administration route and administration method. For example, water, alcohol, edible oil, or the like can be used as the liquid carrier. As the solid carrier, amino acids such as lysine, polysaccharides such as cyclodextrin, organic acid salts such as magnesium stearate, cellulose derivatives such as hydroxylpropylcellulose, and the like can be used.

  The substance selected in the above step (e) further includes isotonic agents, preservatives, wetting agents, emulsifiers, dispersants, stabilizers, solubilizers, excipients, binders, disintegrants, dilutions. Various pharmaceutical additives such as an agent, a buffer, a coloring agent, and a flavoring agent can be blended. In the case of injections, sterilization is performed together with a suitable carrier to obtain a drug.

The agent for preventing or treating human type II diabetes is at least one selected from the group consisting of human fasting blood glucose abnormality, human glucose tolerance abnormality, human insulin resistance, and human lipid abnormality It can be used as a preventive or therapeutic agent for the symptoms.
In addition, the above-mentioned drugs for preventing or treating human type II diabetes are human fasting blood glucose abnormality improving agent, human glucose tolerance lowering inhibitor, human insulin resistance improving agent, human chronic glucose metabolism disorder Also serves as a preventive / therapeutic agent.

  Experiments with mammals must be conducted according to the international principles 3R: Replacement (development of alternative methods), Reduction (reduction of animal numbers), Refinement (reduction of animal pain) (Russell and Burch (1959)). From the ethical point of view, it has been pointed out that screening diabetes drug candidates at the expense of many mammals is problematic.

The silkworm type II diabetes model developed by the present invention is consistent with the idea of Replacement, one of the 3Rs. Also, silkworms are easy to sample blood (body fluids) and measure blood sugar levels, which are difficult for nematodes and Drosophila often used as disease model animals. Therefore, the type II diabetes model silkworm used in the present invention is superior to these small invertebrates as a hyperglycemic model animal.
Furthermore, previous studies have revealed that pharmacokinetics in silkworms is common to mammals (Hamamoto H et al., Comp Biochem Physiol C Toxicol Pharmacol. 2009). Therefore, silkworms are expected to be a new model organism useful as a search system for new antidiabetic drugs.

[Action]
Although the action and principle of screening a candidate substance for preventing or treating human type II diabetes in the steps including the steps (a) to (e) are not clear, the following may be considered.
So far, silkworms have been reported to have a mechanism for maintaining homeostasis of blood glucose levels. In addition, silkworms can store sugar in fat bodies corresponding to muscles and human liver, and have ombibixin, an insulin-like peptide hormone, and downstream of bombyxin, a MAPK signaling pathway similar to that in humans is provided. It has also been reported that insulin signaling pathways exist. Furthermore, recombinant human insulin has an action of enhancing the uptake of sugar in the fat body of silkworm through the activation of PI3 kinase, and by the activation of AMP kinase, which is a pathway other than the insulin signaling pathway, The blood sugar level decreases.

One of the causes of abnormal glucose tolerance (decrease in glucose tolerance) due to chronic feeding of a high-calorie diet in humans is the failure of the blood glucose regulation mechanism due to insulin resistance. It is known that in diabetic mice and diabetic rats, the liver and adipose tissue do not respond to insulin stimulation. As described above, it has been clarified that the blood glucose control mechanism of silkworms has a mechanism similar to that of humans, but there are many unclear points regarding other metabolic control mechanisms.
According to the examples of the present invention, it was found that silkworms that were hyperglycemic by giving a high glucose diet for a certain period showed abnormal fasting blood glucose, decreased glucose tolerance, insulin resistance, and lipid abnormalities at the individual level. These symptoms are consistent with the symptoms of human type II diabetes. As described above, silkworms have similar mechanisms to human blood glucose regulation, and other metabolic regulation mechanisms such as silkworm lipid regulation mechanisms are similar to those of humans. It is done.

[Differences in methods for evaluating substances that lower blood sugar levels in humans using conventional silkworms]
In a conventional evaluation method, a high glucose-containing food is administered to a silkworm for 1 hour, a test substance is administered, and a blood glucose level of the silkworm is measured 6 hours after the test substance is administered. It was evaluated whether or not it was a substance that lowers blood glucose levels (Examples 4, 5, and 10 of Patent Document 1).

The conventional evaluation method has the advantage that the test substance can evaluate a substance that lowers the blood glucose level of a human in a short time, while pioglitazone, which is a therapeutic agent for type II diabetes, evaluates it. (The substance could not be captured (evaluated) as a candidate substance and could not be screened).
The cause of this is thought to be that the hyperglycemia model of silkworms due to short-term intake of high-glucose diet does not simulate the human type II diabetes model.

Therefore, the present invention pays attention to “overeating” which is considered as one of the causes of human type II diabetes, and by giving a high glucose-containing diet for a longer time than the conventional evaluation method, As a result of the examination based on the hypothesis that type II diabetes-like symptoms may appear, human type II diabetes-like symptoms in silkworms fed with high glucose-containing food for a certain period and then fasted for a certain period ( Abnormal fasting blood glucose, glucose tolerance, insulin resistance, lipid abnormalities) were confirmed.
In “the silkworm fed with a high-glucose-containing diet for 1 hour” used in the conventional evaluation method, it was suggested that insulin resistance was not achieved because the blood glucose level was reduced by insulin administration (Patent Document 1). Example 4). Moreover, as a result of measuring the blood glucose level when fasted for 2 hours, 5 hours, and 8 hours after feeding with a high glucose-containing food for 1 hour, the blood glucose level decreased after 2 hours from the start of fasting, and fasting blood glucose abnormality It is suggested that it is not caused (Example 7 of Patent Document 1).
From these results, it is considered that type II diabetes-like symptoms do not appear in “the silkworm fed with a high glucose-containing diet for 1 hour”.

Further, the difference between the evaluation method of the present invention and the conventional evaluation method is the process from the administration of the test substance to the measurement of the blood glucose level. In the conventional evaluation method, as described above, the blood glucose level is measured 6 hours after administration of the test substance, whereas in the present invention, the “step of fasting the silkworm administered with the test substance” is performed. After that, blood sugar level is measured.
The reason for fasting is to measure the fasting blood glucose level. It is known that fasting blood glucose levels are high in human type II diabetics, and this increase in fasting blood glucose levels is a symptom of type II diabetes, such as insulin resistance, glucose metabolism, and abnormal lipid metabolism. It is caused by etc.
The results of an examination based on the hypothesis that silkworms, like humans, can be used to determine whether or not they have type II diabetes-like symptoms by measuring fasting blood glucose levels. The present invention has been completed. Therefore, from the conventional evaluation method, the “step of fasting a silkworm administered with the test substance” cannot be easily conceived.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example and a test example, this invention is not limited to specific ranges, such as a following example.

<Type of silkworm, breeding conditions, injection conditions>
Silkworm fertilized eggs (Hybrid Fuyuyo × Tsukuba Ne) were purchased from Ehime Yoro Co., Ltd. The hatched larvae were given artificial feed silk mate 2S (Nippon Nosan Kogyo Co., Ltd.) at room temperature and grown to 5th instar larvae. As the breeding container, a square type No. 2 petri dish (Eiken Equipment Co., Ltd.) was used from the egg to the 2nd instar larva, and the disposable plastic food pack (Food Pack FD Oduka, Chuo Chemical Co., Ltd.) was used thereafter. The breeding temperature was 27 ° C. Unless otherwise specified, larvae on the first day of the fifth instar, fasted after the fourth instar sleep, were used in the experiments.
The sugar-added sample was prepared by mixing D-Glucose with artificial feed silk mate 2S (Nippon Nosan Kogyo Co., Ltd.) at various weight ratios.
In the silkworm injection experiment, 0.05 mL of a sample was injected into a somatic segment having the pattern of the fifth somatic larvae. The syringe barrel (1 mL) and the injection needle (27G × 3/4) were purchased from Terumo Corporation.

<Method for quantifying blood sugar level in silkworm>
Blood (20 μL) was taken from a scissor cut on the first proleg and mixed with 9 volumes of 0.6 N perchloric acid to precipitate the protein. Centrifugation was performed at 3,000 rpm for 10 minutes, and the supernatant was used as a body fluid extract (hemolymph extract).
The total amount of sugar in silkworm blood was quantified by the phenol sulfate method (Hodge et al). 100 μL of bodily fluid extract diluted to an appropriate concentration with distilled water and 100 μL of 5% (w / v) phenol aqueous solution were mixed, 500 μL of concentrated sulfuric acid was added, and the mixture was vigorously stirred and allowed to stand at room temperature for 20 minutes, followed by absorbance at 490 nm. Was measured. A glucose aqueous solution was used as a standard sugar solution.
Blood glucose concentration was quantified by the glucose oxidase method. 20 μL of body fluid extract diluted to an appropriate concentration with distilled water and 400 μL of enzyme reaction solution (0.12 M Na-phosphate buffer (pH 7.4), 4 units / mL of glucose oxidase, 3 units / mL of peroxidase, 9 mM o-dianisidine) Were mixed and left at room temperature for 40 minutes. Thereafter, 100 μL of 70% concentrated sulfuric acid was added and stirred vigorously, and the absorbance at 530 nm was measured. A glucose aqueous solution was used as a standard sugar solution.

<Determination of triglycerides and free fatty acids in silkworm fat bodies and body fluids>
Silkworms were fed normal food or 10% glucose-containing food for 18 hours. Thereafter, fat bodies and body fluids of silkworms of each group were collected. The amount of triglyceride and free fatty acid in the collected sample was quantified by an enzymatic method. Triglyceride E-Test Wako (Wako Pure Chemicals, Osaka, Japan) was used for triglycerides, and free fatty acids were quantified using NEFA C-Test Wako (Wako Pure Chemicals, Osaka, Japan).

<Reagent>
Recombinant human insulin was purchased from Sigma and dissolved in physiological saline (0.9% NaCl) containing 0.1% acetic acid. Pioglitazone was purchased from LKT Laboratories, Inc. 5 mg of pioglitazone was suspended in 100 μL of 0.1M HCl and boiled for 4 minutes to dissolve. What was vortexed with 900 μL of PBS was used. Metformin (1,1-Dimethylbiguanide Hydrochloride) was purchased from Wako Pure Chemical and dissolved in physiological saline.

<Detection of fat body Akt and JNK phosphorylation>
A fat body excised from a silkworm was washed with Insect saline (130 mM NaCl, 5 mM KCl, 1 mM CaCl ₂ ), and then NP-40 lysis buffer (10 mM Tris / HCl (pH 7.5), 150 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 1% NP-40, 10 mM NaF, 1 mM Na ₃ VO ₄ ) was mixed with 250 μL of solution and sonicated for 5 seconds. Thereafter, TCA precipitation was performed, the protein was subjected to SDS electrophoresis, and transferred to a PVDF membrane. Western blotting using anti-Akt antibody, anti-phosphorylated Akt antibody, anti-JNK antibody and anti-phosphorylated JNK antibody was performed.

<Oil Red O staining of silkworm fat body>
Silkworms were fed normal food or 10% glucose-containing food for 18 hours. Thereafter, fat bodies of silkworms of each group were collected. After washing twice with PBS, it was immersed in a 3.7% formalin solution at room temperature for 30 minutes. After washing twice with PBS, it was immersed in 60% isopropanol for 1 minute, and immersed in Oil Red O staining solution (final concentration 1.8 mg / mL) at room temperature for 20 minutes. Then, it was washed 5 times with a 60% isopropanol solution and photographed. After measuring the weight of the fat body sample from which water had been removed, 200 μL of xylene was added to the fat body and stirred at room temperature for 30 minutes. And it processed for 30 second by OPC (Out put control) 2 mode with the sonicator (Sonifier 450, Branson), and centrifuged for 3 minutes at 10,000 rpm. OD490 of the centrifugal supernatant fraction was measured, and the absorbance per fat body of a constant weight was calculated and compared.

Example 1
[Abnormal fasting blood glucose and decreased glucose tolerance in silkworms that were hyperglycemic by giving glucose for a certain period of time]
Research so far has found that the blood sugar level of silkworms is increased by feeding silkworms with a high glucose diet for 1 hour (Non-patent Document 2). When human insulin was administered to this hyperglycemic silkworm, the blood glucose level decreased, but even when pioglitazone, a therapeutic agent for type II diabetes, was administered, no decrease in blood glucose level was observed. Type II diabetes is caused by reduced glucose tolerance and insulin resistance due to chronic high-calorie diets. Therefore, the conditions under which the glucose tolerance of silkworms decreases and insulin resistance is examined.

  In humans, the criteria for determining that glucose tolerance has decreased include (1) postprandial blood glucose level of 200 mg / dL or higher, (2) fasting blood glucose level of 150 mg / dL or higher, (3) in glucose tolerance test, after glucose load In 30 minutes, the blood glucose level is 200 mg / dL or more. Furthermore, as a criterion for determining insulin resistance, insulin transmission pathway by insulin is not induced (Ryan G. Holzer, et.al., Cell, 2011, 173-184).

  First, we examined whether fasting blood glucose abnormalities occur in silkworms by giving them high-calorie food containing glucose for a certain period of time. A 5 year old silkworm was fed a diet containing 10% (w / w) glucose for 18 hours and then fasted for 24 hours, and then the blood sugar concentration was measured (FIG. 1A). After feeding for 18 hours, the blood glucose level of the high glucose feed group was 2.4 times higher than that of the normal feed group (FIG. 1B). Thereafter, the food was removed, and even after fasting for 24 hours, the high glucose feed group showed a higher blood glucose level than the normal feed group (FIG. 1C). The results in FIG. 1 suggest that fasting blood glucose abnormalities occur in silkworms that have been hyperglycemic by giving a high glucose diet for a certain period of time.

Next, a 5-year-old day 1 silkworm (hereinafter abbreviated as “normal diet-fed silkworm”) fed with a normal diet for 18 hours and a diet containing 10% (w / w) glucose for 18 hours Each of the 5th day old silkworms (hereinafter abbreviated as “high-glucose-feeding silkworms”) was fasted for 24 hours and then subjected to a glucose tolerance test (GTT) (FIG. 2A).
That is, after fasting for 24 hours, 100 mL of a glucose solution (75 mg / mL) was injected into silkworm blood, and then the total sugar concentration and glucose concentration after 0, 15, 30, 60, 90, and 120 minutes were measured.

As a result, it was found that high glucose-feeding silkworms showed higher total sugar concentrations in the blood at 15, 30, 60, 90, and 120 minutes after glucose administration than normal-feeding silkworms ( FIG. 2B). Normally, there is no glucose in the blood of the feeding silkworm, and there is a disaccharide, trehalose. So far, it has been known that glucose and trehalose come to coexist in the body fluid of silkworms when fed with glucose (Non-patent Document 2).
High glucose-fed silkworms showed higher glucose concentrations in body fluids at 15, 30, 60, and 90 minutes after administration of glucose than normal feed-fed silkworms (FIG. 2C).
The result of FIG. 2 suggests that the sugar tolerance is reduced in silkworms that have been hyperglycemic by giving a high glucose diet for a certain period of time.

Example 2
[Enhanced insulin resistance in silkworms fed with high glucose]
One of the causes of a decrease in glucose tolerance due to chronic feeding of a high-calorie diet in humans is the failure of the blood glucose regulation mechanism due to insulin resistance. In diabetic mice and diabetic rats, it is known that the liver and adipose tissue become unresponsive to insulin stimulation (Ryan G. Holzer, et. Al., Cell, 2011, 173-184).
In addition, studies to date have found that addition of insulin to the in vitro tissue culture system of isolated silkworm fat bodies enhances Akt phosphorylation and sugar uptake in fat body cells (non-patented). Reference 2).

  Accordingly, the fat bodies of silkworms that were hyperglycemic by giving glucose feed for a certain period of time and the fat bodies of silkworms that were fed with normal food were extracted, and the response to insulin was compared. That is, for each of the high-glucose-feeding silkworms and the normal-feeding silkworms, fat bodies were removed after 18 hours of feeding, and physiological saline and recombinant human insulin (0.7 mg) were administered to Grace's medium at 27 ° C. After culturing for 3 hours, Western blotting using an anti-phosphorylated Akt antibody and an anti-Akt antibody was performed.

  As a result, Akt phosphorylation was enhanced by insulin in fat bodies extracted from normal feed-fed silkworms, but Akt phosphorylation by insulin was not caused in fat bodies extracted from high-glucose-fed silkworms. It became clear (FIG. 3). This result suggests that silkworm adipocytes that have been hyperglycemic by giving a high glucose diet for a certain period of time have reduced responsiveness to insulin.

Example 3
[Liquid accumulation in blood and fat bodies in silkworms fed with high glucose]
As a cause of insulin resistance in type II diabetic patients, an increase in triglyceride and free fatty acid concentrations in organs such as liver and adipose tissue and blood has been pointed out. In addition, mice that are diabetic by eating a high fat diet (HFD) show symptoms of obesity such as accumulation of triglycerides and glycerol in the liver and an increase in free fatty acids in the blood (Mark J. Czaja). , Trends in Endocrinology and Metabolism, 2010, 707-713). Accumulation of these lipids is thought to cause insulin resistance (Giovanni Tatantino and Armando Caputi, World journal of Gastroenterology, 2011, 3785-3794, Ryan G. Holzer, et. Al., Cell, 2011). 173-184).

  Therefore, it was examined whether lipids accumulated in body fluids and fat bodies of silkworms fed with high glucose. That is, the amounts of triglycerides and free fatty acids in fat bodies and body fluids were quantified after feeding for 18 hours for silkworms fed with normal food and silkworm fed with high glucose.

As a result, when fat droplets of fat body cells were stained with Oil Red O, the fat bodies of high-glucose-fed-feeding silkworms were stained more strongly than the fat bodies of normal-feeding-fed silkworms (FIG. 4A). Furthermore, as a result of extracting the pigment | dye with xylene from the fat body after dyeing | staining process and quantifying the pigment | dye amount, 1.3 times difference was recognized by both (FIG. 4B).
Furthermore, the amount of triglycerides in fat bodies of silkworms fed with high glucose was 1.4 times higher than that of silkworms fed with normal food (FIG. 5A). At this time, there was no significant difference in the amount of free fatty acids in the fat bodies of normal diet-fed silkworms and high glucose feed-fed silkworms (FIG. 5B).

  On the other hand, it is known that the amount of triglycerides and free fatty acids in the blood of patients who are insulin resistant is increased. In addition, studies using mice have also reported that free fatty acids in blood cause insulin resistance. Therefore, it was examined whether the concentrations of triglycerides and free fatty acids were increased in the body fluid of silkworms fed with high glucose. As a result, it was found that the triglyceride and free fatty acid concentrations in the body fluids of silkworms that were hyperglycemic by giving a high glucose diet for a certain period of time were higher than those in the normal diet-fed silkworms (FIGS. 5C and D).

  The above results suggest that silkworms that are hyperglycemic by giving a high glucose diet for a certain period of time exhibit symptoms of lipid abnormalities. In addition, an increase in the amount of triglycerides and free fatty acids in the body fluid suggests that insulin resistance may be induced by the increase in these concentrations.

Example 4
[Enhanced phosphorylation of JNK in fat bodies of silkworm fed with high glucose]
It is known that free unsaturated fatty acids in blood induce insulin resistance by causing phosphorylation of JNK (c-Jun N-terminal kinase) in various tissues (Ryan G. Holzer, et. Al., Cell, 2011, 173-184). As described above, in the silkworm fed with high glucose, the amount of free fatty acid in the blood increased, and the fat body showed insulin resistance.

  Then, next, it was investigated whether the phosphorylation of JNK in a fat body was increasing in this high glucose feed silkworm. The fat bodies of high-glucose-feeding silkworms and normal-feeding silkworms were excised 18 hours after feeding, and the levels of JNK phosphorylation in fat body cells were compared by Western blotting. As a result, it was found that JNK phosphorylation was enhanced in the fat body of this high-glucose-fed-feeding silkworm than that in the normal-fed-feeding silkworm (FIG. 6).

Example 5
[Effect of administration of pioglitazone and metformin on blood glucose level of silkworm fed with high glucose]
An attempt was made to evaluate pioglitazone and metformin, which are drugs for treating type II diabetes, using silkworms fed with high glucose (Cignarelli A., et al Arch Physiol Biochem 2013 early online: 1-11).
For each of the high-glucose-feeding silkworms and the normal-feeding silkworms, the control solution (PBS + 0.01M HCl) or pioglitazone (5 mg / mL) was administered after feeding for 18 hours, and the blood sugar concentration was measured for 24 hours.
In addition, for each of the silkworms fed with high glucose and the silkworm fed with normal food, physiological saline (0.9% NaCl) or metformin (4 mg / mL) was administered after feeding for 18 hours, and the blood sugar concentration for 24 hours Was measured. That is, 0.25 mg of pioglitazone and 0.2 mg of metformin were administered per silkworm (about 1.4 g).
As a result, it was found that when pioglitazone or metformin was administered to high-glucose-fed silkworms, the fasting blood glucose level decreased (FIG. 7).

Test example 1
[Effect of administration of bioglitazone and metformin on the blood sugar level of silkworms used in conventional evaluation methods]
5th day old silkworms were fed a diet supplemented with 10% (w / w) glucose and raised at 27 ° C. for 1 hour, then saline, pioglitazone (0.1 mg / larva), or metformin (0.1 mg / larva) was administered. Blood was collected 6 hours after administration and the sugar concentration was measured.
The results are shown in FIG. Both pioglitazone (FIG. 8A) and metformin (FIG. 8B) did not significantly reduce blood glucose levels compared to when saline was administered.

  From Examples 1-2, it was found that high-glucose-feeding silkworms showed an increase in fasting blood glucose level, a decrease in glucose tolerance at the individual level, and insulin resistance. These symptoms are consistent with the symptoms of human type II diabetes. From Example 3, in this silkworm fed with high glucose, the amount of fat in the fat body and body fluid was increased, and a lipid abnormal (hyperlipidemic) -like symptom was shown. Moreover, from Example 4, the insulin responsiveness in the in vitro tissue culture system was reduced in the fat bodies extracted from silkworms fed with high glucose. Furthermore, from Example 5, when pioglitazone or metformin was administered to this high-glucose feed silkworm, a decrease in blood glucose level was observed. These results suggest that silkworms that have been hyperglycemic by giving glucose for a certain period can be used as a type II diabetes model. The model is expected to be useful for searching for novel type II diabetes therapeutics.

  It has been reported that the intake of a high-calorie diet in mammals increases the amount of triglycerides in the liver and the amount of free fatty acids in the blood. In addition, an increase in blood free fatty acid leads to phosphorylation of JNK, which causes insulin resistance (Ryan G. Holzer, et. Al., Cell, 2011, 173-184, Mark J. Czaja, Trends in Endocrinology and Metabolism, 2010, 707-713, Giovanni Tatantino and Armando Caputi, World journal of Gastroenterology, 2011, 3785-3794). In silkworms, as in mammals, eating a high glucose diet increases the amount of triglycerides in the fat body, and accordingly increases the amount of free fatty acids in the body fluid. It is thought that the free fatty acid induces phosphorylation of Akt in fat body cells, causing insulin resistance, and ultimately causing a decrease in fat body glucose tolerance.

  Most of the sugars in the body fluids of insects such as silkworms and fruit flies are trehalose combined with two molecules of glucose. Insects are thought to have a regulatory mechanism that converts to less toxic trehalose to mitigate the toxicity of glucose. Silkworms are thought to increase the concentration of glucose in body fluids by ingesting excess glucose that exceeds the tolerance of the control mechanism that converts to trehalose.

  According to the present invention, it has been clarified that if a silkworm is fed with a diet containing excessive glucose for a certain period of time, it exhibits human type II diabetes-like symptoms. On the other hand, in mice, in order to show type II diabetes-like symptoms such as an increase in blood glucose level, it is said that it is necessary to give a high fat diet for 2 weeks or more. Therefore, using a silkworm, a type II diabetes model can be constructed in a much shorter time than when using a mouse.

  In addition, the present invention has found that administration of pioglitazone or metformin decreases the blood glucose level during fasting of silkworms that have been given high glucose for a certain period of time to increase hyperglycemia. The present invention is the first example of evaluating the hypoglycemic effect of a therapeutic agent for type II diabetes using invertebrates. Pioglitazone, a thiazolidine derivative, is thought to contribute to the improvement of insulin resistance by enhancing the action of the transcription factor PPARγ (peroxisome proliferator-activated receptor-γ). (Hauner H: The mode of action of thiazolidinediones. Diabetes Metab. Res. Rev., 18: S10-S15, 2002). In mammals, PPARγ is highly expressed in adipocytes and also expressed in skeletal muscle and liver.

Thiazolidine derivative drugs are known to act on various organs and cause a decrease in blood glucose level. Thiazolidine derivative drugs increase the number of insulin-sensitive small fat cells, increase the expression of adiponectin involved in blood glucose regulation, and enhance fatty acid uptake for adipocytes. A thiazolidine derivative induces increased PPARγ expression, sugar uptake, and enhanced glycogen synthesis against skeletal muscle cells. Thiazolidine derivative drugs cause suppression of gluconeogenesis on liver cells.
It has been found that a gene encoding a protein having high homology with PPARγ exists in the silkworm genome. The amino acid sequence of the highly homologous protein is shown in SEQ ID NO: 1. Pioglitazone is presumed to act on various organs of silkworms and cause activation of PPARγ.

  On the other hand, metformin, which is a biguanide, acts on the liver and skeletal muscle of mammals and is thought to contribute to the improvement of diabetic symptoms by activating AMP-activated protein kinase (AMPK) (Zhou G, et al.,: Role of AMP-activated protein kinase in mechanism of metformin action, J. Clin. Invest., 108: 1167-1174, 2001). The present inventors have so far reported that activation of AMPK leads to a decrease in blood glucose level even in silkworms (Non-patent Document 2). Metformin is considered to act on various organs of the silkworm and cause activation of AMPK.

  According to the method for evaluating a therapeutic agent for type II diabetes using the hyperglycemic silkworm of the present invention, not only a novel type II diabetes therapeutic agent can be searched but also fasting blood glucose abnormality, glucose tolerance abnormality, insulin resistance Since it is possible to elucidate the mechanism that causes lipid abnormalities, it is widely used in the field of drug development and the like.

Claims (3)

A method for evaluating whether a test substance is a candidate substance for preventing or treating human type II diabetes,
(A) At least one state selected from the group consisting of abnormal fasting blood glucose, abnormal glucose tolerance, insulin resistance, and lipid abnormality by allowing a silkworm to ingest sugar (A) for at least 18 hours The process of
(B) In the silkworm obtained in the step (a), at least one silkworm selected from the group consisting of abnormal fasting blood glucose, abnormal glucose tolerance, insulin resistance, and lipid abnormality, Administering a test substance;
(C) fasting silkworms administered with the test substance for at least 10 hours ,
(D) a step of measuring the concentration of sugar (B) in the fat body or blood of the fasted silkworm,
A method characterized by comprising: A method for screening a substance for preventing or treating human type II diabetes,
(A) At least one state selected from the group consisting of abnormal fasting blood glucose, abnormal glucose tolerance, insulin resistance, and lipid abnormality by allowing a silkworm to ingest sugar (A) for at least 18 hours The process of
(B) In the silkworm obtained in the step (a), at least one silkworm selected from the group consisting of abnormal fasting blood glucose, abnormal glucose tolerance, insulin resistance, and lipid abnormality, Administering a test substance;
(C) fasting silkworms administered with the test substance for at least 10 hours ,
(D) a step of measuring the concentration of sugar (B) in the fat body or blood of the fasted silkworm,
(E) selecting a substance that reduces the concentration of sugar (B) in the fat body or blood of the silkworm from the test substance,
A method characterized by comprising: A method for producing a prophylactic or therapeutic agent for human type II diabetes,
(A) At least one state selected from the group consisting of abnormal fasting blood glucose, abnormal glucose tolerance, insulin resistance, and lipid abnormality by allowing a silkworm to ingest sugar (A) for at least 18 hours The process of
(B) In the silkworm obtained in the step (a), at least one silkworm selected from the group consisting of abnormal fasting blood glucose, abnormal glucose tolerance, insulin resistance, and lipid abnormality, Administering a test substance;
(C) fasting silkworms administered with the test substance for at least 10 hours ,
(D) a step of measuring the concentration of sugar (B) in the fat body or blood of the fasted silkworm,
(E) selecting a substance that reduces the concentration of sugar (B) in the fat body or blood of the silkworm from the test substance,
(F) mixing the substance selected in step (e) with a pharmaceutically acceptable carrier;
A method characterized by comprising: