JP2005110632A - Model animal for obesity-related diabetes - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a model animal for obesity-related diabetes developing diabetes induced by obesity from a normal animal, an examination method for obesity treating medicine, diabetes prevention medicine and diabetes treating medicine by using the obesity-related diabetes model animal prepared by the method, a method for screening an obesity treating substance, a diabetes prevention substance, a diabetes treating substance, an abnormal sugar metabolism improving substance and a lipid metabolism improving substance and a substance acquired by the screening method. <P>SOLUTION: The model animal is prepared by fattening a hybrid mouse derived from a mouse selected from C57BL/6 mouse, 129/J mouse, MA/J mouse, BALB/c mouse and AKR/J mouse and a mouse selected from DBA/2 mouse, C57BL/KsJ mouse, SWR/J mouse, CBA/LT mouse, C3HeB/FeJ mouse, ICR(CD1) mouse and FVB/NJ mouse by breeding the hybrid mouse with a high-fat feed or excessively feeding the mouse. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for producing an obese diabetes model animal that develops diabetes from obesity, and an obesity therapeutic agent, diabetes preventive agent, diabetes therapeutic agent, and hyperlipidemia therapeutic agent using the obese diabetes model animal produced by the method. It relates to test methods.

In recent years, obesity and type 2 diabetes, whose increase is becoming a major social problem, are closely related. For example, obesity is one of the major risk factors for type 2 diabetes, and it is known to progress from obesity to type 2 diabetes.

  The majority of obesity in humans is caused mainly by lack of exercise and eating a diet with high fat content (hereinafter abbreviated as “high fat diet”), and is not a single genetic disease. On the other hand, most human type 2 diabetes is not a single genetic disease, but it develops due to a decrease in insulin action (insulin resistance) and a decrease in insulin secretion from the pancreas. However, there are many unclear points about the actual mechanism (see, for example, Non-Patent Document 1). And in particular, the cause of the relationship between the two diseases, that is, why obesity is a risk of type 2 diabetes and the cause of progression from obesity to type 2 diabetes has yet to be elucidated.

  Appropriate animal models are needed to elucidate the causes of obesity to type 2 diabetes in humans and to develop therapeutic agents. It is important that the model exhibits a pathology similar to progression from obesity to type 2 diabetes in humans and is free of genetic abnormalities. However, conventionally used diabetes model animals do not develop obesity or diabetes mainly due to dietary obesity, but develop diabetes due to genetic abnormalities. It was not a suitable model animal to elucidate the relationship between

  For example, db / db mice have a mutated leptin receptor gene and develop severe obesity and diabetes almost simultaneously due to abnormal overeating and severe sugar / lipid metabolism disorders (see, for example, Non-Patent Document 2). In addition, KK mice exhibit diabetes symptoms with obesity due to abnormalities in a plurality of specific genes (see, for example, Non-Patent Document 3).

  On the other hand, normal mice have long had inbred strains with various genetic backgrounds, and strains that actually develop diabetes are known due to abnormalities in specific genes related to diabetes. For example, DBA / 2 mice and C57BL / KsJ mice are known to develop severe diabetes when they have a genetic abnormality such as the above-mentioned leptin receptor gene mutation or leptin gene deficiency (eg, non-patented). Reference 4). However, so far, there has been no report that inbred mice develop clear diabetes by forming obesity with a high fat diet.

  Although C57BL / 6 mice raised on a high-fat diet have been reported as showing a pathological condition close to diabetes, C57BL / 6 mice develop insulin resistance due to obesity, but the blood glucose level is mild and diabetes is also Since it does not present, the progress to diabetes is not clear (for example, refer nonpatent literature 5).

  In addition, strains of mice that are likely to become obese by giving a high-fat diet also exist, for example, it has been reported that AKR / J mice form heavier obesity than SWR / J mice by raising high-fat diets (for example, Non-patent documents 6 to 8). However, these strains are not known to develop diabetes due to a high fat diet. Even in cases other than the above examples, there has been no known model mouse that develops obesity due to a high-fat diet that is the main cause of human obesity and further progresses to diabetes. In addition, there has been no report of successful development of obesity / diabetes by breeding a high-fat diet with mice having both the strain of obesity and the strain of diabetes.

Therefore, to date, there is no mouse model that develops diabetes due to obesity caused by a high-fat diet, and an animal model similar to human diabetes is used to analyze the onset mechanism of diabetes, search for therapeutic agents for diabetes, It was difficult to evaluate.
“The Journal of Clinical Endocrinology & Metabolism”, 2001, Vol. 86, p. 4047-4058 “Diabetologia”, 1970, Vol. 6, p. 268-273 “Diabetes & metabolism”, 1997, Volume 23 Suppl 2, p. 38-46 “Diabetes”, 1981, Volume 30, p. 1029-1034 “Metabolism”, 1999, vol. 42, p. 1405-1409 “Journal of Clinical Investigation”, 1994, Vol. 94, p. 1410-1416 “Nature”, 2000, Vol. 404, p. 644-651 “Obesity Research”, 2000, Vol. 8, p. 89-117

  The present invention relates to a method for producing an obese diabetes model animal that develops diabetes from obesity using normal animals, and an obesity therapeutic agent, diabetes preventive agent, and diabetes treatment agent using the obese diabetes model animal produced by the method It is an object of the present invention to provide a screening method for obesity, a substance for treating obesity, a substance for preventing diabetes, a substance for treating diabetes, a substance for improving abnormal sugar metabolism, a substance for improving lipid metabolism, a substance obtained by the screening method, and the like.

The present invention
(1) A method for producing an obese diabetes model mouse comprising the following steps 1) and 2):
1) A mouse selected from C57BL / 6 mouse, 129 / J mouse, MA / J mouse, BALB / c mouse and AKR / J mouse, DBA / 2 mouse, C57BL / KsJ mouse, SWR / J mouse, CBA / Breeding a hybrid mouse with a mouse selected from LT mouse, C3HeB / FeJ mouse, ICR (CD1) mouse and FVB / NJ mouse on a high fat diet;
2) measuring the weight of the mouse of 1) and selecting an obese mouse;
(2) A method for producing an obese diabetes model mouse comprising the following steps 1) and 2):
1) A mouse selected from C57BL / 6 mouse, 129 / J mouse, MA / J mouse, BALB / c mouse and AKR / J mouse, DBA / 2 mouse, C57BL / KsJ mouse, SWR / J mouse, CBA / Breeding a hybrid mouse with a mouse selected from LT mouse, C3HeB / FeJ mouse, ICR (CD1) mouse and FVB / NJ mouse on a high fat diet;
2) Measuring the weight of the mouse of 1) and selecting a mouse whose weight has increased by 20% or more compared to a mouse raised on a normal diet,
(3) A method for producing an obese diabetes model mouse comprising the following steps 1) and 2):
1) A mouse selected from C57BL / 6 mouse, 129 / J mouse, MA / J mouse, BALB / c mouse and AKR / J mouse, DBA / 2 mouse, C57BL / KsJ mouse, SWR / J mouse, CBA / Breeding a hybrid mouse with a mouse selected from LT mouse, C3HeB / FeJ mouse, ICR (CD1) mouse and FVB / NJ mouse on a high fat diet;
2) A step of examining the blood glucose level and / or urine sugar level of the mouse of 1), and selecting a mouse in which an increase in blood glucose level and / or diabetes has been confirmed compared to a mouse raised on a normal diet,
(4) A method for producing obese diabetes model mice comprising the following steps 1) to 3):
1) A mouse selected from C57BL / 6 mouse, 129 / J mouse, MA / J mouse, BALB / c mouse and AKR / J mouse, DBA / 2 mouse, C57BL / KsJ mouse, SWR / J mouse, CBA / Breeding a hybrid mouse with a mouse selected from LT mouse, C3HeB / FeJ mouse, ICR (CD1) mouse and FVB / NJ mouse on a high fat diet;
2) A step of measuring the weight of the mouse of 1) and selecting a mouse whose weight has increased by 20% or more compared to a mouse raised on a normal diet;
3) a step of selecting a mouse in which an increase in blood glucose level and / or diabetes has been confirmed compared to a mouse raised on a normal diet,
(5) A method for producing an obese diabetes model mouse comprising the following steps 1) and 2):
1) A mouse selected from C57BL / 6 mouse, 129 / J mouse, MA / J mouse, BALB / c mouse and AKR / J mouse, DBA / 2 mouse, C57BL / KsJ mouse, SWR / J mouse, CBA / A step of administering and breeding goldthioglucose to a hybrid mouse with a mouse selected from LT mouse, C3HeB / FeJ mouse, ICR (CD1) mouse and FVB / NJ mouse;
2) measuring the weight of the mouse of 1) and selecting an obese mouse;
(6) A method for producing an obese diabetic model mouse comprising the following steps 1) and 2):
1) A mouse selected from C57BL / 6 mouse, 129 / J mouse, MA / J mouse, BALB / c mouse and AKR / J mouse, DBA / 2 mouse, C57BL / KsJ mouse, SWR / J mouse, CBA / Administering a gold thioglucose to a hybrid mouse with a mouse selected from LT mouse, C3HeB / FeJ mouse, ICR (CD1) mouse and FVB / NJ mouse;
2) measuring the body weight of the mouse of 1), and selecting a mouse whose body weight has increased by 20% or more compared to a mouse not administered with goldthioglucose;
(7) A method for producing an obese diabetic model mouse comprising the following steps 1) and 2):
1) A mouse selected from C57BL / 6 mouse, 129 / J mouse, MA / J mouse, BALB / c mouse and AKR / J mouse, DBA / 2 mouse, C57BL / KsJ mouse, SWR / J mouse, CBA / Administering a gold thioglucose to a hybrid mouse with a mouse selected from LT mouse, C3HeB / FeJ mouse, ICR (CD1) mouse and FVB / NJ mouse;
2) Examining the blood glucose level and / or urine sugar level of the mouse of 1), and selecting a mouse in which an increase in blood glucose level and / or diabetes has been confirmed compared to a mouse to which goldthioglucose was not administered,
(8) A method for producing an obese diabetes model mouse comprising the following steps 1) to 3):
1) A mouse selected from C57BL / 6 mouse, 129 / J mouse, MA / J mouse, BALB / c mouse and AKR / J mouse, DBA / 2 mouse, C57BL / KsJ mouse, SWR / J mouse, CBA / Administering a gold thioglucose to a hybrid mouse with a mouse selected from LT mouse, C3HeB / FeJ mouse, ICR (CD1) mouse and FVB / NJ mouse;
2) A step of measuring the body weight of the mouse of 1) and selecting a mouse having a body weight increased by 20% or more compared to a mouse not administered with goldthioglucose;
3) a step of selecting a mouse in which an increase in blood glucose level and / or diabetes has been confirmed compared to a mouse to which goldthioglucose has not been administered,
(9) selected from (1) to (8), wherein the hybrid mouse is an F1 hybrid of C57BL / 6 and DBA / 2 mice [(C57BL / 6 x DBA / 2): F1] A method for producing an obese diabetes model mouse according to any one of the above,
(10) An obese diabetes model mouse produced by the production method according to any one of (1) to (9),
(11) A method for testing a prophylactic and / or ameliorating substance for at least one disease selected from abnormal sugar metabolism, obesity and abnormal lipid metabolism, comprising the following steps 1) and 2):
1) a step of administering a test substance to the obese diabetes model mouse according to (10);
2) Body weight, blood glucose level, urinary glucose level, blood insulin level, blood glucose level and insulin level in glucose tolerance test, blood triglyceride concentration, insulin content or insulin secretory capacity of islets of Langerhans and insulin Measuring at least one selected from reactivity,
(12) A method for testing a prophylactic and / or ameliorating substance for at least one disease selected from abnormal sugar metabolism, obesity and abnormal lipid metabolism, comprising the following steps 1) and 2):
1) A step of contacting a test substance with a tissue derived from the obese diabetes model mouse according to (10);
2) a step of measuring at least one selected from triglyceride content, insulin content, insulin secretion ability and insulin responsiveness in the tissue of 1),
(13) A test method for a prophylactic and / or ameliorating substance for at least one disease selected from abnormal sugar metabolism, obesity and abnormal lipid metabolism, comprising the following steps 1) and 2):
1) a step of administering a test substance to the obese diabetes model mouse according to (10);
2) a step of measuring at least one selected from triglyceride content, glucose uptake ability and glucose synthesis ability in the tissue removed from the mouse of 1),
(14) A method for testing a prophylactic and / or ameliorating substance for at least one disease selected from abnormal sugar metabolism, obesity and abnormal lipid metabolism, comprising the following steps 1) and 2):
1) a step of administering a test substance to the obese diabetes model mouse according to (10);
2) A step of measuring at least one selected from triglyceride content, glucose uptake ability and glucose synthesis ability in the tissue removed from the mouse of 1);
3) selecting a test substance in which at least any one selected from a decrease in triglyceride content, an increase in insulin content, an increase in insulin secretion ability, and an increase in insulin reactivity is observed;
(15) A method for testing a prophylactic and / or ameliorating substance for at least one disease selected from abnormal glucose metabolism, obesity and abnormal lipid metabolism, comprising the following steps 1) to 3):
1) a step of administering a test substance to the obese diabetes model mouse according to (10);
2) Body weight, blood glucose level, urine sugar level, blood insulin level, blood glucose level and insulin level in glucose tolerance test, blood triglyceride concentration, insulin content of Langerhans Island, Langerhans Island Measuring at least one selected from insulin secretory ability and insulin responsiveness;
3) Decrease in body weight, decrease in blood glucose level, decrease in urine sugar level, increase in blood insulin concentration, decrease in blood glucose level or insulin level in glucose tolerance test, decrease in blood triglyceride concentration, Langerhans Island Selecting a test substance in which at least any one selected from an increase in insulin content, an increase in insulin secretory ability of islets of Langerhans and an increase in insulin responsiveness is observed,
(16) A test method for a prophylactic and / or ameliorating substance for at least one disease selected from abnormal sugar metabolism, obesity and abnormal lipid metabolism, comprising the following steps 1) to 3):
1) A step of contacting a test substance with a tissue derived from the obese diabetes model mouse according to (10);
2) a step of measuring at least one selected from triglyceride content, insulin content, insulin secretion ability and insulin reactivity in the tissue of 1);
3) selecting a test substance in which at least any one selected from a decrease in triglyceride content, an increase in insulin content, an increase in insulin secretion ability, and an increase in insulin reactivity is observed;
(17) A method for testing a prophylactic and / or ameliorating substance for at least one disease selected from abnormal sugar metabolism, obesity and abnormal lipid metabolism, comprising the following steps 1) to 2):
1) A mouse selected from C57BL / 6 mouse, 129 / J mouse, MA / J mouse, BALB / c mouse and AKR / J mouse, DBA / 2 mouse, C57BL / KsJ mouse, SWR / J mouse, CBA / Administering a test substance to a hybrid mouse with a mouse selected from an LT mouse, a C3HeB / FeJ mouse, an ICR (CD1) mouse and an FVB / NJ mouse, and rearing them on a high fat diet;
2) Body weight, blood glucose level, urinary glucose level, blood insulin level, blood glucose level and insulin level in glucose tolerance test, blood triglyceride concentration, insulin content or insulin secretory capacity of islets of Langerhans and insulin Measuring at least one selected from reactivity,
(18) A test method for a prophylactic and / or ameliorating substance for at least one disease selected from abnormal sugar metabolism, obesity and abnormal lipid metabolism, comprising the following steps 1) to 3):
1) A mouse selected from C57BL / 6 mouse, 129 / J mouse, MA / J mouse, BALB / c mouse and AKR / J mouse, DBA / 2 mouse, C57BL / KsJ mouse, SWR / J mouse, CBA / Administering a test substance to a hybrid mouse with a mouse selected from an LT mouse, a C3HeB / FeJ mouse, an ICR (CD1) mouse and an FVB / NJ mouse, and rearing them on a high fat diet;
2) Body weight, blood glucose level, urine glucose level, blood insulin level, blood glucose level and insulin level in glucose tolerance test, blood triglyceride concentration, insulin content of Langerhans island, insulin secretion of Langerhans island Measuring at least one selected from potency and insulin responsiveness;
3) Decrease in body weight, decrease in blood glucose level, decrease in urine sugar level, increase in blood insulin concentration, decrease in blood glucose level or insulin level in glucose tolerance test, decrease in blood triglyceride concentration, Langerhans Island Selecting a test substance in which at least any one selected from an increase in insulin content, an increase in insulin secretory ability of islets of Langerhans and an increase in insulin responsiveness is observed,
(19) A test method for a prophylactic and / or ameliorating substance for at least one disease selected from abnormal sugar metabolism, obesity and abnormal lipid metabolism, comprising the following steps 1) to 3):
1) A step of administering a test substance to a mouse of F1 hybrid [(C57BL / 6 x DBA / 2): F1] of a C57BL / 6 mouse and a DBA / 2 mouse and rearing them on a high fat diet;
2) Body weight, blood glucose level, urine glucose level, blood insulin level, blood glucose level and insulin level in glucose tolerance test, blood triglyceride concentration, insulin content of Langerhans island, insulin secretion of Langerhans island Measuring at least one selected from potency and insulin responsiveness;
3) Decrease in body weight, decrease in blood glucose level, decrease in urine sugar level, increase in blood insulin concentration, decrease in blood glucose level or insulin level in glucose tolerance test, decrease in blood triglyceride concentration, Langerhans Island Selecting a test substance in which at least any one selected from an increase in insulin content, an increase in insulin secretory ability of islets of Langerhans and an increase in insulin responsiveness is observed,
(20) A method for testing a prophylactic and / or ameliorating substance for at least one disease selected from abnormal sugar metabolism, obesity and abnormal lipid metabolism, comprising the following steps 1) to 2):
1) A mouse selected from C57BL / 6 mouse, 129 / J mouse, MA / J mouse, BALB / c mouse and AKR / J mouse, DBA / 2 mouse, C57BL / KsJ mouse, SWR / J mouse, CBA / Administering a gold thioglucose and a test substance to a hybrid mouse with a mouse selected from an LT mouse, a C3HeB / FeJ mouse, an ICR (CD1) mouse and an FVB / NJ mouse;
2) Body weight, blood glucose level, urinary glucose level, blood insulin level, blood glucose level and insulin level in glucose tolerance test, blood triglyceride concentration, insulin content or insulin secretory capacity of islets of Langerhans and insulin Measuring at least one selected from reactivity,
(21) A method for testing a prophylactic and / or ameliorating substance for at least one disease selected from abnormal sugar metabolism, obesity and abnormal lipid metabolism, comprising the following steps 1) to 3):
1) A mouse selected from C57BL / 6 mouse, 129 / J mouse, MA / J mouse, BALB / c mouse and AKR / J mouse, DBA / 2 mouse, C57BL / KsJ mouse, SWR / J mouse, CBA / Administering a gold thioglucose and a test substance to a hybrid mouse with a mouse selected from an LT mouse, a C3HeB / FeJ mouse, an ICR (CD1) mouse and an FVB / NJ mouse;
2) Body weight, blood glucose level, urine glucose level, blood insulin level, blood glucose level and insulin level in glucose tolerance test, blood triglyceride concentration, insulin content of Langerhans island, insulin secretion of Langerhans island Measuring at least one selected from potency and insulin responsiveness;
3) Decrease in body weight, decrease in blood glucose level, decrease in urine sugar level, increase in blood insulin concentration, decrease in blood glucose level or insulin level in glucose tolerance test, decrease in blood triglyceride concentration, Langerhans Island Selecting a test substance in which at least any one selected from an increase in insulin content, an increase in insulin secretory ability of islets of Langerhans and an increase in insulin responsiveness is observed,
(22) The hybrid mouse is an F1 hybrid [(C57BL / 6 x DBA / 2): F1] of C57BL / 6 mouse and DBA / 2 mouse, and is selected from (17) to (21) A test method for a prophylactic and / or ameliorating substance for at least one disease selected from the abnormal sugar metabolism, obesity and abnormal lipid metabolism according to any one of the above:
(23) In the prophylactic and / or ameliorating substance for at least one disease selected from abnormal sugar metabolism, obesity and abnormal lipid metabolism obtained by the method of (11) to (22) (24) (23) A therapeutic agent for at least one disease selected from diabetes, obesity, and hyperlipidemia, comprising the substance described above as an active ingredient,
Consists of.

  The present inventors have found that in C57BL / 6 mice, the formation of obesity when fed a high fat diet is more prominent than in DBA / 2 mice and C57BL / KsJ mice. In addition, the present inventors are an F1 hybrid of C57BL / 6, a strain having a trait that is likely to form obesity, and DBA / 2, a strain having a trait that is likely to cause diabetes but is difficult to obese. By using BDF1 mice, we succeeded in developing severe diabetes after normal mice were obese with a high fat diet.

  The present inventors have bred BDF1 mice, which are widely used as normal mice, under high-fat diet conditions, so that the mice can exhibit (1) insulin resistance and increased insulin secretion 8 weeks after feeding the high-fat diet. To develop simple obesity with or without diabetes, (2) After 12 weeks of feeding, in addition to obesity, cause glucose intolerance, marked increase in blood glucose level, appearance of urine sugar, (3) Langerhans Insulin insulin content decreased, insulin secretion ability by glucose stimulation in isolated islets of Langerhans decreased, and (4) discovered that there are abnormalities in insulin staining and glucagon staining in islets of Langerhans.・ Successfully distinguishing and developing type 2 diabetes, these pathologies are similar to progression from obesity to type 2 diabetes in humans, and human type 2 diabetes patients We succeeded in reproducing the exhaustion of the islets of Langerhans suggested in.

  The present inventors further revealed that in other strain mice (C57BL / 6), which are widely used, insulin resistance obesity develops in the same high fat diet, but diabetes does not develop. We found that the genetic factors necessary to develop diabetes from diet-induced obesity exist in DBA / 2 mice.

  Furthermore, the present inventors further used an obesity diabetes model mouse produced by the above production method, a method for testing an obesity therapeutic agent, a diabetes preventive agent, and a diabetes therapeutic agent, as well as a substance for treating obesity, a substance for preventing diabetes, and diabetes. The present invention has succeeded in providing a screening method for therapeutic substances, substances that improve glucose metabolism abnormalities, and substances that improve lipid metabolism, whereby obesity therapeutic drugs, diabetes preventive drugs, and diabetes therapeutic drugs, obesity therapeutic substances, diabetes preventive substances, and It has been shown that a substance for improving diabetes, a substance for improving abnormal sugar metabolism, and a substance for improving lipid metabolism can be provided, thereby completing the present invention.

  The present invention provides a method for producing an obese diabetes model animal that develops diabetes from obesity using a normal animal. In addition, an obesity treatment drug, a diabetes prevention drug, and a test method for a diabetes treatment drug using the obese diabetes model animal produced by the method are provided. Furthermore, a substance having an obesity treatment effect using the obesity diabetes model animal produced by the method, a substance having a diabetes prevention effect, and / or a screening method for a substance having a diabetes treatment effect, and a substance obtained by the screening method Was provided.

  In the present specification, the “characters that tend to become obesity” refers to the traits that form obesity when a high-fat diet is ingested. Specifically, as a mouse having a trait that tends to become obesity, specifically, C57BL / 6 Mention may be made of mice, 129 / J mice, MA / J mice and AKR / J mice. Further, in the present specification, “a trait that is likely to cause diabetes when obese” refers to a trait that is correlated with obesity and diabetes, and as a mouse having a trait that is likely to cause diabetes when obese Mice that develop diabetes when they have a leptin receptor mutation (db / db) or gold thioglucos treatment (eg DBA / 2, C57BL / KsJ, SWR / J, CBA / LT, C3HeB / FeJ, ICR (CD1) and FVB / NJ mice).

  In the present specification, the “obese diabetic model mouse” refers to a mouse that develops diabetes after being obese by the method of the present invention.

1. Animal In the present invention, an animal used for producing an animal that develops diabetes is not particularly limited as long as it is a mouse having both a trait that tends to cause obesity and a trait that tends to cause diabetes when obese, for example, , C57BL / 6 mice, 129 / J mice, MA / J mice, BALB / c mice and AKR / J mice, DBA / 2 mice, C57BL / KsJ mice, SWR / J mice, CBA / LT A mouse hybrid selected from a mouse, a C3HeB / FeJ mouse, an ICR (CD1) mouse, and an FVB / NJ mouse can be mentioned, and an F1 hybrid of a C57BL / 6 mouse and a DBA / 2 mouse is preferable [(C57BL / 6 x DBA / 2): F1], particularly preferably F1 hybrids of female C57BL / 6 and male DBA / 2 mice [(C57BL / 6 x DBA / 2): F1 ] Male BDF1 mice (Nippon Charles River). Here, 129 / J mice and AKR / J mice are strains that form obesity with a high fat diet. In addition, C57BL / KsJ mice, SWR / J mice, CBA / LT mice, C3HeB / FeJ mice and FVB / NJ mice have leptin receptor mutations (db / db) or are treated with gold thioglucos for diabetes. It is a strain that develops. In addition, a recombinant inbred mouse (for example, a BXD mouse which is a DBA / 2 recombinant inbred line with C57BL / 6) by a combination of these mice can also be used.

  As a control group of mice that do not develop diabetes, for example, C57BL / 6 mice (Nippon Charles River) can be used. DBA / 2 mice (Nippon Charles River) and C57BL / KsJ mice (Claire Japan) can be used as animals that are not obese due to a high fat diet. The above mice can be purchased from, for example, Japan Charles River and Japan Clare, but can also be obtained from other breeding facilities, and further, self-breeding using the purchased mice and mice established through self-breeding can also be used. I can do it.

2. Feed The feed used for breeding mice is not particularly limited as long as it can be used normally for breeding mice. In the present specification, the “normal diet” is not particularly limited as long as it is a feed that can be normally used as a mouse feed. For example, commercially available feed for rodent rearing (FR-2 pellet, Funabashi Farm: Moisture, crude protein, crude fat, ash, and soluble nitrogen-free substances can be used in the following composition: 8.0, 20.8, 4.8, 3.2, 5.0, 58.2%) it can.

  In the present specification, the “high fat diet” refers to a diet having a high fat content compared to a normal diet, and can be prepared, for example, by adding fat to a normal diet. The amount of fat added to a normal diet is not particularly limited as long as it can cause diabetes, but preferably the proportion of fat is 10% to 70% by weight with respect to the total weight of the feed including fat. %, More preferably 20% to 50%, and still more preferably 25%.

  The type of fat to be added is not particularly limited. For example, lard, animal oils other than lard (for example, fish oil such as beef tallow, tuna oil, sardine oil) and vegetable oil (for example, coconut oil, rapeseed oil, palm oil, soybean oil, Sesame oil, safflower oil, cottonseed oil, etc.). Moreover, as long as it contains fat, a food containing fat can be used as it is, or unrefined crude oil, partially refined oil, or completely refined oil can be used as appropriate. These fats may be a single type or a mixture of a plurality of fats. A high-fat diet is not limited to a mixture of normal diet and fat, but a normal diet and fat are prepared separately, and the intake of both diets increases as a result of the intake of both mice. Good. An example of a high fat diet is a pasty feed made by adding lard in an amount of 25% of the final weight of FR-2 powder (Funabashi Farm).

  As described above, as a high-fat diet, in addition to the method of calculating and preparing the amount of fat based on the fat content in the feed, by increasing the proportion of fat in the total calorie intake Can also be prepared. Examples of high fat diets based on calorie intake include rodent breeding diets with lard added at a concentration of 45 calories in the diet, but the type and concentration of fat is limited to this It is not a thing.

  Other diets that induce obesity and insulin resistance (eg, diets with high sucrose content; Surwit RS et al., Metabolism, 44: 645-651, 1995) should also be used as ingredients in diets that form obesity. Can do.

3. Breeding method (1) Formation of obesity by high fat diet Mice can be raised by methods well known to those skilled in the art.

  Before giving a high-fat diet, it is desirable to keep a regular diet for a certain period of time (referred to as “pre-breeding”). There is no particular limitation on the period of pre-breeding, but it is usually about one week. However, pre-breeding is not necessary.

  The age of the mouse used in the experiment is not particularly limited as long as it causes diabetes by giving a high-fat diet, and can be selected appropriately depending on the type of mouse used, but female C57BL / 6 mice and male DBA / 2 When male F1 hybrid [(C57BL / 6 x DBA / 2): F1] male BDF1 mouse (Nippon Charles River) is used, for example, 3 weeks to 12 weeks, preferably 5 weeks to 10 weeks Weekly mice, more preferably 7-week-old mice can be used.

  Onset of diabetes can be confirmed by breeding with a high-fat diet for mice that have been pre-bred. A high-fat diet can be taken in a certain amount or can be taken freely. In addition, a certain amount of water can be ingested or can be ingested freely. Mouse food intake and / or water intake can also be recorded as needed.

  Moreover, it can be reared by a method in which a normal diet and a high fat diet are given at a certain ratio without making all diets a high fat diet. As such a method, for example, after being raised on a high-fat diet for 6 days, it is raised on a normal diet for 1 day, again on a high-fat diet for 6 days, and on a normal diet for 1 day. Such cases may be mentioned, but the method is not particularly limited as long as obesity, abnormal blood glucose level, and / or onset of diabetes can be confirmed.

  Moreover, it is preferable to prepare a mouse reared on a normal diet as a control.

  The breeding period is not particularly limited as long as obesity, abnormal blood glucose level, and / or diabetes can be confirmed. For example, when male BDF1 mice are used, 5 to 20 weeks, preferably 7 to 15 weeks, Preferably it can be 12 weeks.

  In addition, about the breeding period of a mouse | mouth, you may determine the breeding period not according to the number of breeding days but according to another standard such as terminating the breeding when the body weight reaches a certain weight.

(2) Formation of obesity by goldthioglucose In addition to the method described in (1) above, a method of administering goldthioglucose can also be used as a method for obese mice. In mice given goldthioglucose, overeating (Debons AF et al., Fed Proc, 36: 143-147, 1977) leads to obesity.

  Goldthioglucose is administered intraperitoneally at a dose of 0.4 to 1.5 g / kg of mouse body weight.

  The feed given to the mouse may be a normal diet or a high fat diet, and other breeding conditions are in accordance with (1).

4). Obesity and Diabetes Assay (1) Confirmation of mouse obesity formation Obesity formation in mice is measured by measuring body weight or adipose tissue weight (eg epididymal fat, mesenteric fat, groin fat and / or brown fat). Can be done.

  For example, in the case of a mouse raised on a high-fat diet, when (a) the body weight is increased by 20% or more, (b) the body weight is increased by 5 g or more, compared with a control mouse raised on a normal diet. If (c) mesenteric fat and / or groin subcutaneous fat is increased by 30% or more, (d) mesenteric fat and / or groin subcutaneous fat is increased by 300 mg or more, It can be confirmed that the mouse has become obese when it corresponds to at least one of (a) to (d).

(2) Pathological analysis The pathological condition of mice bred with a high fat diet can be examined by the results of various measurements shown below. It is preferable to comprehensively judge from these measurement results whether or not mice bred with a high fat diet have abnormal sugar metabolism.

(A) Insulin resistance The pathological analysis of obesity that has developed insulin resistance can be performed by measuring blood hormones (for example, those that have been pointed out to be associated with obesity such as insulin and leptin). These measurement samples can be obtained by methods commonly used by those skilled in the art. When blood insulin and blood leptin are statistically significantly increased compared to control animals, it can be determined that there is an obesity condition that has developed insulin resistance.

(B) Diabetes Diabetes can be assayed by quantifying blood or urine glucose concentration and hemoglobin glycation rate (HbA1c). Since the unit of glucose concentration in plasma and urine is generally mg / dl (where dl is 100 ml), it was followed. As a result of the measurement, if the plasma glucose concentration is statistically significantly higher than that of the control animal, it can be determined that there is an abnormality in glucose metabolism, the plasma glucose concentration exceeds 250 mg / dl, and the urine glucose concentration is If it exceeds 50 mg / dl, it can be determined that diabetes has occurred. In addition, when HbA1c is significantly higher than that of the control animal, it can be determined that the diabetic state continues constantly.

  A mouse that has developed diabetes by the method of the present invention is referred to as an obese diabetes model mouse.

(C) Blood lipid Blood lipid can be changed by measuring plasma total cholesterol concentration, plasma free fatty acid concentration, and plasma neutral fat concentration. These concentrations can be measured by a method commonly used by those skilled in the art, but can also be measured, for example, by the following method. The total cholesterol concentration in plasma can be measured using cholesterol CII-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). Plasma free fatty acid concentration can be measured using NEFA C-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). Plasma neutral fat concentration can be measured using GPO-TRINDER (SIGMA).

(D) Glucose tolerance Glucose tolerance can be examined by oral glucose tolerance test (Perley MJ and Kipnis DM, J Clin Invest, 46: 1954-1962, 1967). In mice bred with a high fat diet, a rapid increase in blood glucose level, for example, exceeding 500 mg / dl, and subsequent sustained hyperglycemic state are observed after oral glucose load.

(E) Insulin content in islets of Langerhans Insulin content in islets of Langerhans is determined by isolating islets of Langerhans using existing methods (Walters GH et al., Horm Metab Res Suppl, 25: 20-26, 1991). Insulin can be measured using an ultrasensitive rat insulin measurement kit (Seikagaku Corporation). The difference in the size of the islets of Langerhans can be determined by measuring the DNA content of the used islets of Langerhans (for example, PicoGreen DNA quantitation kit, Molecular Probe).

  Insulin concentration in the islets of Langerhans is reduced by more than 20% compared to control animals in mice fed with a high fat diet and having abnormal sugar metabolism.

(F) Insulin secretion test Insulin secretion test in isolated islets of Langerhans is performed by suspending isolated islets of Langerhans in a buffer solution of physiological salt concentration (for example, Krebs-Ringer bicarbonate buffer). Thus, insulin that is stimulated by different glucose concentrations and released into the supernatant can be determined by measuring by the method described above. In mice raised on a high-fat diet and showing abnormal glucose metabolism, insulin secretion in response to glucose is significantly reduced. In some cases, a decrease in the maximum insulin secretion ability induced by stimulation with high potassium chloride may be observed.

(G) Immunohistochemical analysis of islets of Langerhans After immunohistochemical analysis of islets of Langerhans, the excised pancreas was fixed using, for example, Buan solution (saturated picric acid: formalin: acetic acid = 15: 5: 1) This can be carried out by embedding in paraffin according to a conventional method, deparaffinizing a sliced section, and immunostaining according to a conventional method. Anti-insulin antibodies (for example, H-86, Santa Cruz) and anti-glucagon antibodies (Zymed) can be used, but other antibodies against peptides existing on the islets of Langerhans (for example, somatostatin) can also be used. In mice raised on a high-fat diet and abnormal sugar metabolism, in addition to decreased insulin staining, glucagon-positive cells originally localized in the outer periphery of Langerhans Island appear inside Langerhans Island. Structural changes are observed.

(3) Predicting the onset of diabetes Based on the confirmation of obesity formation in mice in (1) above and the measurement results of pathological conditions in (2), whether or not to develop diabetes is measured by the degree of obesity, for example, body weight or adipose tissue weight It became clear that it can be predicted. For example, when male BDF1 mice are used, it can be predicted that diabetes occurs when the body weight is increased by 20% or more compared to a normal diet or when the body weight exceeds 48 g. In this way, obese diabetes model mice can be obtained.

5). Analysis of the expression level of polynucleotides in diabetic mice As described in the item “4.” above, mice having both a trait that tends to cause obesity and a trait that tends to cause diabetes when obese are ingested a high-fat diet Or obese diabetes model mice can be produced by eating goldthioglucose and overeating. Abnormal glucose metabolism such as diabetes in humans is often caused by ingestion and / or overeating of a high-fat diet, and mice produced according to the present invention have a mechanism of glucose metabolism similar to that of human glucose metabolism. An abnormality has been caused. Therefore, the mouse shown in the present invention can be used to elucidate the cause of diabetes and the cause of progression from obesity to diabetes.

  In addition, animals with abnormal sugar metabolism, such as diabetes, often have abnormal lipid metabolism. Therefore, the mouse obtained by the present invention is an effective model animal for elucidating the mechanism of human sugar metabolism abnormality and / or lipid metabolism abnormality. Experiments for elucidating the onset mechanism of abnormal sugar metabolism and / or abnormal lipid metabolism typified by diabetes can be performed by combining experimental methods commonly performed by those skilled in the art. For example, the following methods can also be used. It can be carried out.

(1) Method for analyzing a polynucleotide whose expression is fluctuating A polynucleotide whose expression level is different between a normal mouse and a mouse with diabetes is a gene associated with abnormal sugar metabolism and / or abnormal lipid metabolism. Can be determined to be coded. Such a gene can be specifically identified by the following method.

(I) Breeding and selection of mice From a mouse fed with a high fat diet and a mouse fed with a normal diet according to the method described in the above section “3. Or, take a sample after the body weight has reached a certain standard.

  That is, in the present invention, not only analysis of mice that have already developed diabetes due to intake of a high fat diet, but also intake of a high fat diet, increasing body weight, is expected to develop diabetes in the future. A mouse can also be used.

  When breeding mice, mice were bred with normal diet and were of standard weight, mice that were bred with normal diet and became obese, mice that were bred with high-fat diet, Samples can also be analyzed by dividing them into groups of mice that have been fed on a fat diet but have not become obese with normal weight. At the same time, according to the method described in the section of “4. Assay method for obesity and diabetes” for each mouse as needed, the body weight adipose tissue weight, glucose concentration in blood and urine, glycation rate of hemoglobin, At least one item selected from the measurement of plasma total cholesterol concentration, plasma free fatty acid concentration, and plasma neutral fat concentration is measured, and the state of each mouse individual such as glucose metabolism is examined.

(Ii) Preparation of total RNA In order to analyze the expression level of a polynucleotide in a mouse, it is generally desirable to analyze the difference in the expression level of a polynucleotide in RNA derived from a specific tissue. As long as it can be analyzed, RNA derived from all tissues can also be used.

  The tissue from which RNA is obtained is not particularly limited, and examples thereof include blood, pancreas, small intestine, liver, muscle, and adipose tissue containing white adipocytes. Among these, when using mouse white adipose tissue Is shown below. When extracting total RNA from mouse white adipose tissue, cut out the white adipose tissue, taking care not to contaminate other tissues after sacrifice and blood collection, and remove the tissue from the RNA extraction solvent (eg phenol or other ribonuclease). It is preferable to dissolve directly in a component containing a component having an inactivating action. Or, scrape the cells carefully with a scraper so as not to destroy the cells of the tissue, or gently extract the cells from the tissue using a proteolytic enzyme such as trypsin. To the RNA extraction step.

  RNA extraction methods include guanidine thiocyanate / cesium chloride ultracentrifugation, guanidine thiocyanate / hot phenol method, guanidine hydrochloride method, acidic guanidine thiocyanate / phenol / chloroform method (Chomczynski, P. and Sacchi, N., ( 1987) Anal.Biochem., 162, 156-159) can be employed, but the acidic guanidine thiocyanate / phenol / chloroform method is preferred. Commercially available reagents for RNA extraction (for example, ISOGEN (manufactured by Nippon Gene Co., Ltd.), TRIZOL reagent (manufactured by Gibco BRL)) and the like can also be used according to the protocol attached to the reagent.

  It is preferable that the obtained total RNA is further purified to mRNA alone if necessary. Although the purification method is not particularly limited, it is known that most mRNAs present in the cytoplasm of eukaryotic cells have a poly (A) sequence at the 3 ′ end. By adsorbing mRNA to the oligo (dT) probe, capturing the mRNA on the paramagnetic particles immobilized with streptavidin using the binding between biotin / streptavidin, washing and then eluting the mRNA. MRNA can be purified. Alternatively, a method may be employed in which mRNA is adsorbed on an oligo (dT) cellulose column and then purified by elution. Furthermore, mRNA can be further fractionated by sucrose density gradient centrifugation or the like. However, for the method of the present invention, the purification step of these mRNAs is not essential, and as long as the expression of the polynucleotide having the nucleotide sequence described in 1) above can be detected, the total RNA is converted into the subsequent steps. It can also be used.

(Iii) A step of identifying a polynucleotide having a difference in expression level between mice The expression level of the polynucleotide is prepared by preparing cRNA or cDNA from the total RNA obtained in (ii) above, and using an appropriate label. By labeling, the signal intensity can be detected.

  Hereinafter, as an analysis method of the expression level of polynucleotide, an analysis method using a solid phase sample and some other analysis methods will be described.

(A) Analysis method using solid phase sample a-1) Solid phase sample Examples of the solid phase sample include the following.

a-1-1) Gene chip:
A gene chip on which an antisense oligonucleotide synthesized based on an EST (expressed sequence tag) sequence or mRNA sequence on a database is immobilized can be used. As such a gene chip, a gene chip (Lipshutz, RJ et al. (1999) Nature genet. 21, supplement, 20-24) manufactured by Affymetrix can be used. It is not limited, You may manufacture based on a well-known method. Most preferably, the EST sequence or mRNA sequence that forms the base of the antisense oligonucleotide that is immobilized on the gene chip is derived from the same animal as the animal or animal cell to be analyzed. When analyzing mRNA derived from mouse, mouse-derived mRNA is most preferable. For example, Affymetrix mouse 19K set (Murine 19K SET: 19KA, 19KB, 19KC) and mouse 11K set (Murine 11K SET: 11KA, 11KB) Can be used.

b) Array or membrane filter on which a cDNA or RT-PCR product prepared from mouse total RNA or total RNA obtained from a specific tissue is immobilized:
The cDNA or RT-PCR product is cloned by performing reverse transcriptase reaction or PCR with primers prepared based on sequence information such as mouse EST database. Further, commercially available arrays and filters (for example, Intelligene: manufactured by Takara Bio Inc.) may be used, and the above cDNA and RT-PCR products may be used with a commercially available spotter (for example, GMS417 Arrayer: Takara Bio). It may be produced by solid-phase using a product manufactured by Co., Ltd.

a-1-2) Preparation and analysis of probe As a labeled probe, not a specific mRNA clone but a labeled all expressed mRNA is used. Although unpurified mRNA may be used as a starting material for probe preparation, it is desirable to use poly (A) + RNA purified by the method described above. Below, the preparation method and detection / analysis method of a labeled probe when various solid-phased samples are used will be described.

a-2) Affymetrix gene chip:
A biotin-labeled cRNA probe is prepared according to the protocol (Affymetrix expression analysis technical manual) attached to the gene chip manufactured by Affymetrix. Next, according to the protocol (expression analysis technical manual) attached to the gene chip manufactured by Affymetrix, hybridization and analysis are performed using an analysis device (GeneChip Fluidics Station 400) manufactured by Affymetrix, and luminescence by avidin is detected and analyzed. Do.

a-3) Array:
When preparing cDNA from poly (A) ⁺ RNA by reverse transcriptase reaction, it is necessary to label the cDNA so that the cDNA can be detected. When labeling with a fluorescent dye, For example, d-UTP labeled with Cy3, Cy5, etc.) is added to fluorescently label cDNA. In this case, be used as a mixture of both the if labeled test substance added cell derived poly (A) ⁺ and RNA to control cell-derived poly (A) ⁺ RNA with different dyes, after hybridization when it can. For example, when a commercially available array of Takara Bio Inc. is used as the array, hybridization and washing are performed according to the protocol of the company, and a fluorescence signal detector (for example, GMS418 array scanner (manufactured by Takara Bio Inc.)) is used. Analysis is performed after detecting the fluorescent signal. However, the array to be used is not limited to a commercially available array, and may be a homemade or specially made array.

a-4) Membrane filter:
When cDNA is prepared from poly (A) ⁺ RNA using reverse transcriptase, a labeled probe is prepared by adding a radioisotope (for example, d-CTP or the like), and hybridization is performed by a conventional method. After hybridization and washing using an Atlas system (Clontech), which is a filter microarray, detection and analysis are performed using an analysis device (for example, Atlas Image: Clontech).

  In any of the methods a) to c), each mouse white adipocyte-derived probe is hybridized with a normal mouse white adipocyte-derived probe. At this time, the conditions other than the hybridization other than the probe to be used are the same. In the case of using fluorescently labeled probes, if each probe is labeled with a different fluorescent dye, a mixture of both probes can be hybridized to a single immobilized sample at a time to read the fluorescence intensity (Brown, P. O. et al. (1999) Nature genet, 21, supplement, 33-37).

(B) Other analysis methods As analysis methods other than the above, the subtraction cloning method (see Experimental Medicine separate volume, New Genetic Engineering Handbook, Yodosha (1996) P32-35), Differential Display Method (Basic Biochemistry Experimental Method 4 Gene Experiment II. Application, Tokyo Kagaku Doujin (2001), p125-128) can be mentioned, but other methods may be used as long as the difference in gene expression level can be analyzed.

(Iv) Evaluation / Determination As a result of the above analysis, polynucleotides with significantly different expression levels in normal mice and mice-derived white adipocytes having abnormal sugar metabolism can be identified as genes associated with abnormal sugar metabolism. Here, the expression level is “substantially different” means that, for example, using an Affymetrix gene chip, Affymetrix microarray Suite Ver. When analyzed using 3.0, the average difference value is increased or decreased by a factor of two.

(2) Search for homologous polynucleotides Polynucleotides that can be presumed to have similar functions by conducting homology searches with known databases for polynucleotides found by the method of (1). Can be identified. The polynucleotide function identified by homology search can also elucidate the function of the polynucleotide by examining specific expression by the above method. Homologous polynucleotides can be searched not only between homologous organisms but also between other species. The search for homologous polynucleotides can be performed on data registered in a database such as GenBank using a search program such as BLAST, but can also be searched using commercially available search software. The polynucleotide having the sequence thus searched can be obtained by performing a PCR reaction using an appropriate PCR primer using a cDNA library derived from each organism.

(3) Sugar metabolism-related polynucleotide In addition to the polynucleotide specified in (1) and (2) above, it hybridizes with each polynucleotide under stringent conditions and has abnormal sugar metabolism with normal mice. Polynucleotides that differ in expression from mice can also be included in the polynucleotides of the present invention.

(4) Assay for Insulin Resistance-improving Agent Sensitive Polynucleotide The expression level of the polynucleotide obtained as described above varies significantly between normal mice and mice with abnormal sugar metabolism in mouse-derived white adipocytes. It is a polynucleotide that affects changes in sugar metabolism and / or lipid metabolism, and is effective in screening for substances that affect sugar metabolism and / or lipid metabolism, including insulin resistance improvers.

  First, full-length cDNA is obtained from a mouse or human cDNA library according to a known method such as colony hybridization using the identified polynucleotide fragment as a probe. This full-length cDNA is introduced into mouse or human cells and highly expressed to examine whether sugar metabolism and / or lipid metabolism are affected.

  In order to express cDNA in an animal individual, a method of incorporating the obtained full-length cDNA into a viral vector and administering it to an animal can be mentioned. Examples of the gene introduction method using a viral vector include a method of introducing cDNA into a DNA virus such as a retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, or RNA virus. . Of these, methods using retroviruses, adenoviruses, adeno-associated viruses, and vaccinia viruses are preferred.

  Non-viral gene transfer methods include a method in which an expression plasmid is directly administered intramuscularly (DNA vaccine method), a liposome method, a lipofection method, a microinjection method, a calcium phosphate method, an electroporation method, etc. The DNA vaccine method and the liposome method are preferred.

  In addition, for cultured cells, full-length cDNA is converted into cells with insulin sensitivity, such as muscle cells derived from humans, mice, rats, etc., hepatocytes, adipocytes, or cells that differentiate into muscle cells, hepatocytes, adipocytes. Introduction, high expression, and examination of the effects of each target cell on the functions of each target cell, specifically, the functions that regulate glycolipid metabolism such as sugar and lipid production and uptake, or glycogen accumulation can do. Conversely, an antisense nucleic acid against the total RNA of the gene to be tested can be introduced into a cell to examine how it affects the function of each target cell. Another method for suppressing gene expression is to use a double-stranded single-stranded RNA (siRNA) (“Jeans and Developments”), 2001. January 15, Volume 15, Issue 2, p. 188-200). For example, siRNA can be introduced according to methods described in the literature, and the expression level and glucose concentration of the gene targeted by siRNA can be examined.

In introducing a full-length cDNA into an animal or cell, the cDNA is incorporated into a vector containing an appropriate promoter and a sequence involved in expression, and a host cell is transformed with the vector. As a vertebrate cell expression promoter, a promoter that is usually located upstream of the gene to be expressed, an RNA splice site, a polyadenylation site, a transcription termination sequence, and the like can be used. You may have. Examples of the expression vectors include pSV2dhfr (Subramani, S. et al. (1981) Mol. Cell. Biol. 1, 854-864) having the SV40 early promoter, retroviral vectors pLNCX, pLNSX, pLXIN, pSIR ( Clontech), cosmid vector pAxCw (Takara Bio), and the like, but are not limited thereto. The expression vector includes diethylaminoethyl (DEAE) -dextran method (Luthman, H. and Magnusson, G. (1983) Nucleic Acids Res., 11, 1295-1308), calcium phosphate-DNA coprecipitation method (Graham, FL). And van der Eb, AJ (1973) Virology 52, 456-457), and electric pulse perforation (Neumann, E. et al. (1982) EMBO J. 1, 841-845). Dihydrofolate reductase-deficient strains of COS cells (Gluzman, Y. (1981) Cell 23, 175-182, ATCC: CRL-1650) and Chinese hamster ovary cells (CHO cells, ATCC: CCL-61). rlaub, G. and Chasin, LA (1980) Proc. Natl. Acad. Sci. USA 77, 4126-4220), human fetal kidney-derived 293 cells (ATCC: CRL 1573), etc. It is not limited to these. Thus, a desired transformant can be obtained.
It is also possible to produce a knockout animal by genetic manipulation and examine what effect appears on the function of regulating sugar metabolism and / or lipid metabolism. Conversely, in animals that exhibit symptoms such as obesity and diabetes, it is also possible to produce a transgenic animal so that the target gene is highly expressed, and to examine whether the pathological condition is improved or treated. The transgenic animal can be obtained by obtaining a fertilized egg from the animal, transplanting the gene into a pseudopregnant animal after gene transfer, and generating it. The procedure is known in the art (Development Engineering Experiment Manual (Tatsuji Nomura) Supervision, edited by Motoya Katsaki, published in 1987) and Japanese Patent Laid-Open No. 5-48093). Specifically, for example, in the case of a mouse, an ovulation inducing agent is first administered to a female mouse, then mated with a male of the same strain, and the pronuclear fertilized egg is collected from the oviduct of the female mouse the next day. Next, the DNA fragment solution to be introduced is injected into the pronucleus of the fertilized egg using a micro glass tube. In addition, regulatory genes such as promoters and enhancers for expressing the introduced gene in animal cells are not particularly limited as long as they function in the introduced animal cell. A fertilized egg into which DNA has been injected is transplanted into the oviduct of a pseudopregnant foster mother mouse (Slc: ICR, etc.), and is born by natural delivery or caesarean section approximately 20 days later.

  As a method for confirming that the animal thus obtained holds the transgene, for example, DNA is extracted from the tail of the animal, and the DNA is used for specific sense and antisense primers. PCR amplification method, digesting the DNA with a restriction enzyme, gel electrophoresis, blotting the DNA in the gel on a nylon membrane, etc., and then performing Southern blot analysis using all or part of the labeled transgene as a probe The method etc. can be mentioned.

6). Analysis of polypeptide expression levels in obese diabetic model mice Judge that polypeptides that differ in expression levels between normal mice and obese diabetic model mice are related to abnormal glucose metabolism and / or abnormal lipid metabolism be able to. Such a polypeptide can be specifically identified by the following method.

(1) Preparation of sample As the sample, blood, pancreas, small intestine, liver, muscle, and mouse derived from the same as described in the section of “5. Analysis of expression level of polynucleotide in diabetic mouse” above. Adipose tissue containing white adipocytes is preferred. The sample is used after removing insoluble substances by high-speed centrifugation as necessary. For example, polyacrylamide electrophoresis of polypeptides derived from normal mice and mice in which abnormal sugar metabolism and / or abnormal lipid metabolism are observed, identifies polypeptides that differ in their expression levels, and uses an amino acid sequencer, etc. The amino acid sequence is determined and the polypeptide is identified based on the amino acid sequence information. On the other hand, it is already known that there is a relationship with sugar metabolism and / or lipid metabolism, and the expression level of the predicted polypeptide can be examined using ELISA / RIA or the like.

  As a sample for ELISA / RIA, for example, a mouse-derived sample is used as it is or a sample appropriately diluted with a buffer solution is used. For the sample for Western blotting (for electrophoresis), for example, an extract of adipose tissue containing serum, pancreas, small intestine, liver, muscle, and white adipocytes is used as it is, or appropriately diluted with a buffer, and SDS is used. -Mix with sample buffer solution (manufactured by Sigma, etc.) containing 2-mercatol ethanol for polyacrylamide electrophoresis. In the case of dot / slot blotting, for example, using a blotting apparatus, the collected serum, the pancreas, small intestine, liver, muscle, adipose tissue itself containing white adipocytes, or those diluted appropriately with a buffer solution, etc. Adsorb directly to the membrane.

(2) Immobilization of the sample In order to specifically detect the polypeptide in the sample obtained as described above, the sample is precipitated by immunoprecipitation, a method using ligand binding, etc. Detection can be performed without immobilization, or the sample to be detected as it is can be immobilized. In the case of immobilizing a polypeptide, as a membrane used for Western blotting, dot blotting or slot blotting, a nitrocellulose membrane (for example, manufactured by Bio-Rad), a nylon membrane (for example, High Bond-ECL (for example) Amersham Pharmacia, etc.), cotton membranes (for example, blot absorbent filters (Biorad), etc.) or polyvinylidene difluoride (PVDF) membranes (for example, BioRad, etc.).

  As a so-called blotting method for transferring a polypeptide from a gel after electrophoresis to a membrane, a wet blotting method (CURRENT PROTOCOLS IN IMMUNOLOGY volume 2 ed by JE Coligan, AM Kruisbeek, DH Margulies, EM Shevach, W. Strober), semi-dry Formula blotting (see CURRENT PROTOCOLS IN IMMUNOLOGY volume 2 above) and the like. Equipment for dot blotting and slot blotting is also commercially available (for example, Bio-dot).

  On the other hand, in order to perform detection and quantification by the ELISA method / RIA method, a sample or a diluted solution thereof (for example, 0.05% azide) is placed on a dedicated 96-well plate (for example, immunoplate maxi soap (manufactured by Nunk)). The inner bottom surface of the well by placing phosphate buffered saline (so-called “PBS”) containing sodium chloride and allowing it to stand at 4 ° C. to room temperature overnight or at 37 ° C. for 1 to 3 hours. The polypeptide is adsorbed on to immobilize.

(3) Antibody The antibody used specifically recognizes the polypeptide to be detected and / or quantified or a part thereof. Such antibodies can include, for example, antibodies that bind to any specific polypeptide but do not bind to any other mouse or human derived protein.

  The antibody may be obtained by using a conventional method (for example, Shinsei Kagaku Kogaku Kenkyu 1, Protein 1, p.389-397, 1992) to the animal as an antigen protein or an arbitrary polypeptide selected from its amino acid sequence. It can be obtained by immunizing and collecting and purifying antibodies produced in vivo. In addition, according to known methods (for example, Kohler and Milstein, Nature 256, 495-497, 1975, Kennet, R. ed., Monoclonal Antibody p. 365-367, 1980, Prenum Press, NY) A hybridoma can be established by fusing an antibody-producing cell that produces an antibody against a sex-improving agent-sensitive polypeptide and a myeloma cell to obtain a monoclonal antibody.

(4) Measurement and determination In the above method, the detection results are compared between a sample collected from an obese diabetic model mouse and a sample collected from a normal mouse. Peptides can be determined to be associated with abnormal sugar metabolism and / or abnormal lipid metabolism.

7). Evaluation of Glucose Metabolism Improvement Substance Obesity Diabetes Model Mice Obtained by the Present Invention are antidiabetic, antidiabetic, antihyperlipidemic, antihyperlipidemic, obesity, antiobesity, abnormal glucose metabolism This is effective for elucidating and evaluating the action of an improving substance and / or a lipid metabolism improving substance, and for testing, evaluating and / or screening for a sugar metabolism abnormality improving substance and / or a lipid metabolism improving substance. Hereinafter, a therapeutic drug for diabetes, a preventive drug for diabetes, a therapeutic drug for hyperlipidemia, a preventive drug for hyperlipidemia, a therapeutic drug for obesity, a preventive drug for obesity, a substance that improves glucose metabolism abnormality and / or a substance that improves lipid metabolism. The test method, evaluation method and / or screening method will be described.

(1) Test substance The “test substance” used in the present invention is already known as a diabetes therapeutic drug, diabetes preventive drug, hyperlipidemia therapeutic drug, hyperlipidemia preventive drug, obesity therapeutic drug, obesity preventive drug. Any substance that is known to have an effect of improving abnormal sugar metabolism and / or abnormal lipid metabolism, and a substance that does not have this effect can be used. In the following, a description will be given separately for substances whose sugar metabolism abnormality improving effect is already known (sugar metabolism abnormality improving substance) and substances whose effect is not yet known.

(1-1) Substance for improving sugar metabolism abnormality The substance for improving sugar metabolism abnormality used in the present invention may be any substance having a function of improving sugar metabolism abnormality, and examples thereof include an insulin resistance improving agent. . The insulin resistance improving agent refers to a drug used for the treatment of diabetes, and examples of the insulin resistance improving agent include a pharmaceutical composition having an insulin resistance improving agent as an active ingredient. `` Insulin resistance improving agent '' is a drug that improves insulin resistance through an action mechanism that increases insulin receptor sensitivity by enhancing insulin receptor signaling and compensates for deficiency, and is used as an agent for diabetes The As such an insulin resistance improving agent, for example,
Troglitazone (5- [4- (6-hydroxy-2,5,7,8-tetramethylchroman-2-ylmethoxy) benzyl] -2,4-thiazolidinedione) described in JP-A-60-051189 and Pharmacologically acceptable salts thereof (hydrochlorides, etc.)
Pioglitazone (5- [4- [2- (5-ethyl-pyridine-) described in JP-A-61-267580 (EP Patent No. 193,256 or US Pat. No. 4,687,777)) 2-yl) ethoxy] benzyl] -2,4-thiazolidinedione) and pharmacologically acceptable salts thereof (hydrochlorides, etc.),
Rosiglitazone (5- [4- [2- (N-methyl-N-2-) described in JP-A-9-512249 (WO95 / 21608 or US Pat. No. 5,002,953)) Pyridylamino) ethoxy] benzyl] -2,4-thiazolidinedione) and pharmacologically acceptable salts thereof (maleic acid salt, etc.),
GI-262570 described in WO 00/8002 (chemical name is 2 (S)-(2-benzoylphenylamino) -3- [4- [2- (5-methyl-2-phenyloxazole-4- Yl) ethoxy] phenyl] propionic acid) and pharmacologically acceptable salts thereof (hydrochlorides, etc.)
JTT-501 (4- [4- [2- (5-methyl-2-phenyl) described in WO95 / 18125 (EP publication 684,242 or US Pat. No. 5,728,720)) -Oxazol-4-yl) ethoxy] benzyl-3,5-isoxazolidinedione) and pharmacologically acceptable salts thereof (hydrochloride, etc.)
AZ-242 (2-ethoxy-3- [4- [2- (4) described in WO99 / 62872 (EP Publication No. 1,084,103 or US Pat. No. 6,258,850)) -Methylsulfonyloxyphenyl) ethoxy] phenyl] propanoic acid) and pharmacologically acceptable salts thereof (such as sodium salt),
MCC-555 (5- [6- (2-fluorobenzyloxy) naphthalene-] described in JP-A-6-247945 (EP Publication No. 604,983 or US Pat. No. 5,594,016) 2-ylmethyl] thiazolidine-2,4-dione) and pharmacologically acceptable salts thereof,
YM-440 ((Z) -1,4-bis [4- (3,5) described in WO94 / 25448 (EP Patent 696,585 or US Pat. No. 5,643,931)) -Dioxo-1,2,4-oxadiazolidin-2-ylmethyl] phenoxy] -2-butene) and pharmacologically acceptable salts thereof,
KRP-297 (5- (2,4-dioxothiazolidine-5-ylmethyl) -2-methoxy-N- (4) described in JP-A-10-87641 (US Pat. No. 5,948,803). -Trifluoromethylbenzyl) benzamide) and pharmacologically acceptable salts thereof,
T-174 (5- [2- (2-naphthylmethyl) -5-benz] described in JP-A No. 64-56675 (EP Patent No. 283035 or US Pat. No. 4,897,393) Oxazolylmethyl] -2,4-thiazolidinedione) and pharmacologically acceptable salts (hydrochloride, etc.),
NC-2100 (5- (7-benzyloxy-3-quinoylmethyl) -2,4 described in JP-A-9-100200 (EP Publication No. 787725 or US Pat. No. 5,693,651) -Thiazolidinedione) and pharmacologically acceptable salts thereof (hydrochloride, etc.),
NN-622 ((S) -3- [4- [2- (phenoxazin-10-yl) ethoxy] phenyl] -2 described in WO99 / 19313 (US Pat. No. 6,054,453) -Ethoxypropanoic acid) and pharmacologically acceptable salts thereof (such as sodium salts),
BMS-298585 (N- (4-methoxyphenoxycarbonyl) -N- [4- [2- (5-methyl-2-phenyl-4-oxazolyl) ethoxy] benzyl] glycine) described in WO011 / 21602 And pharmacologically acceptable salts thereof (hydrochlorides, etc.),
5- [4- (6-Methoxy-1-methylbenzimidazol-2-ylmethoxy) benzyl] thiazolidine-2,4-dione and pharmacologically acceptable salts thereof (hydrochlorides) described in EP Publication No. 745600 etc),
Oxazole compounds, oxadiazolidine compounds, thiazolidine compounds, or phenoxazine compounds.

(1-2) Substance whose sugar metabolism abnormality improvement effect is not known About the substance whose sugar metabolism abnormality improvement effect is not known, the improvement method of sugar metabolism abnormality can be investigated with the following method. Examples of substances to be examined for the effect include compounds, microbial metabolites, extracts of plants and animal tissues, derivatives thereof or mixtures thereof. It is also possible to use nucleic acids or derivatives thereof (including antisense oligonucleotides, ribozymes, RNAi, etc.) designed to increase the expression level of specific genes.

(2) Mice, tissues or cells derived from the mice According to the method described in the above section “3. Breeding method”, the mice used in this experiment are not only obese diabetic model mice that have already developed diabetes, but are still diabetic. Mice that do not develop can also be used.

  If the test substance is administered and not administered using a mouse that has already developed diabetes, if the abnormal glucose metabolism and / or abnormal lipid metabolism are improved, the test substance is abnormal glucose metabolism and / or Or it can be judged that it has the improvement effect of lipid metabolism abnormality.

  In addition, when the test substance is administered compared to the case where the test substance is administered simultaneously with the case where the test substance is administered at the same time when the mouse that does not develop diabetes is bred with a high fat diet or administered with gold thioglucose If no obesity, abnormal glucose metabolism and / or abnormal lipid metabolism are observed, the test substance can be a preventive substance for obesity, abnormal glucose metabolism and / or abnormal lipid metabolism.

  These include a method in which a test substance is administered to an individual mouse and then the body weight of the animal individual is measured, the expression level of the polynucleotide and / or the expression level of the polypeptide in the organ or tissue cell extracted from the animal individual Blood glucose and urine glucose concentration, hemoglobin glycation rate, plasma total cholesterol concentration, plasma free fatty acid concentration, plasma neutral fat concentration, blood insulin concentration, blood leptin concentration, and blood It can be examined by any of the methods for examining at least one selected from adiponectin concentration or a combination of these methods. Here, the test substance refers to the substance described in (1-1) or (1-2) above. The specimen is not particularly limited as long as it is blood or an organ or tissue to be analyzed, but is preferably at least selected from blood, pancreas, small intestine, liver, muscle, and adipose tissue containing white adipocytes. Any one is included.

  Extraction of total RNA derived from a mouse, mouse tissue or mouse-derived cell, measurement of the expression level of a polynucleotide, and measurement of the expression level of a polypeptide can be performed according to the methods described above.

(3) Influence of Test Substance on Sugar Metabolism and / or Lipid Metabolism Hereinafter, the case where a mouse individual is used and the case where a mouse-derived tissue is used will be described, respectively, but the present invention affects the sugar metabolism and / or lipid metabolism of the test substance. However, the method is not limited to these methods as long as it can be examined.

  Detection of polynucleotide in this method, measurement of polynucleotide expression level, detection of polypeptide, measurement of polypeptide expression level, measurement of insulin concentration, glucose concentration, total cholesterol concentration, free fatty acid concentration, and neutral fat concentration Can be performed by the method described above.

  In addition, it is known that the polypeptide and / or polynucleotide whose expression level is to be measured in the following method is related to sugar metabolism and / or lipid metabolism, and changes in expression level and sugar metabolism and / or It is preferable to use one that has been found to be related to the effect of improving lipid metabolism. Examples of such substances include glucose 6-phosphatase, fructose-1,6-bisphosphatase, pyruvate carboxylase or phosphoenolpyruvate carboxykinase, which are rate-limiting enzymes for gluconeogenesis, and glucose transporters involved in sugar uptake. Glycerol phosphate acyltransferase or diacylglycerol acyltransferase involved in neutral fat synthesis, acyl Co-A synthase, acyl Co-A carboxylase or fatty acid synthase involved in fatty acid synthesis, lipoprotein lipase involved in lipolysis, hormone-sensitive lipase And carnitine palmitoyltransferase or acyl-Co-A oxidase involved in fatty acid oxidation, and TNF-α, a hormone released from adipose tissue. Alternatively, adiponectin, a protein such as an insulin receptor or insulin receptor substrate involved in insulin signal transmission, and / or a gene thereof can be mentioned, but are not limited thereto.

  Hereinafter, test method and evaluation method for diabetes therapeutic agent, diabetes preventive agent, hyperlipidemia therapeutic agent, hyperlipidemia preventive agent, obesity therapeutic agent, obesity preventive agent, glucose metabolism abnormality improving substance and / or lipid metabolism improving substance The screening method will be specifically described.

A) When using a mouse individual A-1) Including the following steps 1) and 2):
1) a step of administering a test substance to an obese diabetes model mouse;
2) At least one selected from the body weight, blood glucose level, blood insulin level, blood glucose level in the oral glucose tolerance test, blood triglyceride concentration, and the insulin content or insulin secretory capacity of the islets of Langerhans The process of measuring one.

A-2) including the following steps 1) to 2):
1) A step of contacting a test substance with a tissue derived from an obese diabetes model mouse;
2) A step of measuring at least one selected from a triglyceride content, an insulin content, and an insulin secretion ability in the tissue of 1).

A-3) including the following steps 1) and 2):
1) a step of administering a test substance to an obese diabetes model mouse;
2) From the body weight, blood glucose level, blood insulin level, blood glucose level in oral glucose tolerance test, blood triglyceride concentration, insulin content of Langerhans island, and insulin secretory ability of Langerhans island of the mice administered the test substance Measuring at least one selected.

A-4) including the following steps 1) and 2):
1) A step of contacting a test substance with a tissue derived from an obese diabetes model mouse;
2) A step of measuring at least one selected from the triglyceride content, the insulin content and the insulin secretory ability in the tissue of 1).

A-5) including the following steps 1) and 3):
1) a step of administering a test substance to an obese diabetes model mouse;
2) At least one selected from the body weight, blood glucose level, blood insulin level, blood triglyceride concentration, insulin content of islets of Langerhans, and insulin secretory capacity of islets of mice administered with the test substance Measuring step;
3) A step of selecting a test substance in which at least one selected from a decrease in body weight, a decrease in blood glucose level, an increase in blood insulin concentration, and a decrease in blood neutral fat concentration is observed.

A-6) including the following steps 1) to 2):
1) A mouse selected from C57BL / 6 mouse, 129 / J mouse, MA / J mouse, BALB / c mouse and AKR / J mouse, DBA / 2 mouse, C57BL / KsJ mouse, SWR / J mouse, CBA / Administering a test substance to a mouse selected from LT mice, C3HeB / FeJ mice, ICR (CD1) mice and FVB / NJ mice and rearing them on a high fat diet;
2) At least one selected from the body weight, blood glucose level, blood insulin level, blood glucose level in the oral glucose tolerance test, blood triglyceride concentration, and the insulin content or insulin secretory capacity of the islets of Langerhans The process of measuring one.

A-7) including the following steps 1) to 3):
1) A mouse selected from C57BL / 6 mouse, 129 / J mouse, MA / J mouse, BALB / c mouse and AKR / J mouse, DBA / 2 mouse, C57BL / KsJ mouse, SWR / J mouse, CBA / Administering a test substance to a mouse selected from LT mice, C3HeB / FeJ mice, ICR (CD1) mice and FVB / NJ mice and rearing them on a high fat diet;
2) Selected from 1) mouse body weight, blood glucose level, blood insulin level, blood glucose level in oral glucose tolerance test, blood triglyceride concentration, insulin content of islets of Langerhans, and insulin secretory capacity of islets of Langerhans Measuring at least one of them.
3) A step of selecting a test substance in which at least one selected from a decrease in body weight, a decrease in blood glucose level, an increase in blood insulin concentration, and a decrease in blood neutral fat concentration is observed.

A-8) The following steps 1) to 3) are included.
1) A step of extracting total RNA from a sample collected from a mouse administered with a test substance;
2) A step of detecting a difference in the expression level of the polynucleotide between the total RNA derived from 1) and the total RNA obtained from a sample collected from an animal not administered with the test substance;
3) A step of analyzing the difference in the expression level of the polynucleotide according to 2) to determine the effect of improving abnormalities in sugar metabolism and / or abnormal lipid metabolism of the test substance.

A-9) including the following steps 1) and 2):
1) a step of detecting the expression level of a polypeptide in a sample collected from a mammal individual administered with a test substance using an antibody or a ligand that specifically binds to the polypeptide;
2) Analyzing the difference between the expression level of the polypeptide detected in 1) and the expression level of the polypeptide in a sample collected from a mammal individual who was not administered the test substance, and the insulin resistance of the present invention of the test substance Determining the effect of promoting or suppressing the function of the sex improving agent-sensitive polypeptide.

A-10) including the following steps 1) to 3):
1) a step of immobilizing all polypeptides in a sample collected from a mammal individual administered with a test substance;
2) a step of detecting the expression level of the polypeptide in the solid phased polypeptide using an antibody or a ligand that specifically binds to the polypeptide;
3) Analyzing the difference between the expression level of the polypeptide detected in 2) and the expression level of the polypeptide in a sample collected from a mammal individual not administered the test substance, and improving the insulin resistance of the present invention A step of determining the effect of promoting or suppressing the function of the agent-sensitive polypeptide.

B) When using mouse tissue B-1) Including the following steps 1) to 4):
1) A step of bringing a test substance into contact with a tissue derived from a mouse that has developed diabetes;
2) a step of measuring at least one selected from triglyceride content, insulin content and insulin secretory capacity in the tissue of 1);
3) A step of selecting a test substance in which at least one selected from a decrease in triglyceride content, a decrease in insulin content, and an increase in insulin secretion ability is observed.

B-2) The following steps 1) to 3) are included.
1) A step of bringing a test substance into contact with a mouse-derived tissue;
2) A step of extracting total RNA from the tissue of 1);
3) a step of detecting a difference in the expression level of the polynucleotide between the total RNA derived from 2) and the total RNA obtained from a mouse tissue not brought into contact with the test substance;
3) A step of analyzing the difference in the expression level of the polynucleotide according to 2) to determine the effect of improving abnormalities in sugar metabolism and / or abnormal lipid metabolism of the test substance.

B-3) including the following steps 1) and 2):
1) A step of bringing a test substance into contact with a mouse-derived tissue;
2) a step of detecting the expression level of the polypeptide in the tissue contacted with the test substance using an antibody or a ligand that specifically binds to the polypeptide;
3) By analyzing the difference between the expression level of the polypeptide measured in 2) and the expression level of the polypeptide in the mouse-derived tissue that was not contacted with the test substance, abnormal glucose metabolism and / or abnormal lipid metabolism of the test substance A step of determining an improvement effect.

B-4) including the following steps 1) to 3):
1) A step of bringing a test substance into contact with a mouse-derived tissue;
2) The step of immobilizing all polypeptides derived from the tissue of 1);
3) a step of detecting the expression level of the polypeptide in the solid phased polypeptide using an antibody or a ligand that specifically binds to the polypeptide;
4) Analyzing the difference between the expression level of the polypeptide detected in 3) and the expression level of the polypeptide in the mouse-derived tissue that was not brought into contact with the test substance, abnormal glucose metabolism and / or abnormal lipid metabolism of the test substance A step of determining an improvement effect.

(4) Evaluation The test method can examine the improvement effect and / or prevention effect of the test substance against obesity, abnormal sugar metabolism and / or abnormal lipid metabolism. Substances for which improvement and / or prevention effects on obesity, abnormal sugar metabolism and / or abnormal lipid metabolism are found by this method are antidiabetic agents, antidiabetic agents, antihyperlipidemic agents, antihyperlipidemic agents , A therapeutic agent for obesity, a preventive agent for obesity, a substance that improves glucose metabolism abnormality and / or a substance that improves lipid metabolism.

8). Glucose metabolism abnormality and / or lipid metabolism abnormality improvement agent and preventive agent Obesity, abnormal glucose metabolism and / or obtained by the method of the present invention by the method described in the item “7. Alternatively, a substance having an improving effect and / or a preventive effect on abnormal lipid metabolism may be used as a therapeutic and / or prophylactic agent for lipid metabolism disorders such as diabetes and / or hyperlipidemia or obesity. When used as, per se or mixed with appropriate pharmacologically acceptable excipients, diluents, etc., orally by tablets, capsules, granules, powders or syrups, or It can be administered parenterally by injection, suppository, patch, or external preparation.

  These formulations include excipients (eg sugar derivatives such as lactose, sucrose, sucrose, mannitol, sorbitol; starch derivatives such as corn starch, potato starch, alpha starch, dextrin; cellulose derivatives such as crystalline cellulose; Gum arabic; dextran; organic excipients such as pullulan; and silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate and magnesium metasilicate aluminate; phosphates such as calcium hydrogen phosphate; Carbonates such as calcium; inorganic excipients such as sulfates such as calcium sulfate; and lubricants (eg, stearic acid, calcium stearate, metal stearate such as magnesium stearate) Salt; talc; colloidal silica; bead wax; Waxes such as wax; boric acid; adipic acid; sulfate such as sodium sulfate; glycol; fumaric acid; sodium benzoate; DL leucine; lauryl sulfate such as sodium lauryl sulfate and magnesium lauryl sulfate; Silicic acids such as silicic acid hydrate; and the above-mentioned starch derivatives), binders (for example, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, and the same as the above-mentioned excipients) And disintegrants (for example, cellulose derivatives such as low-substituted hydroxypropylcellulose, carboxymethylcellulose, carboxymethylcellulose calcium, internally crosslinked sodium carboxymethylcellulose; And starch, celluloses chemically modified such as sodium carboxymethyl starch and cross-linked polyvinyl pyrrolidone), emulsifiers (eg, colloidal clays such as bentonite and bee gum; magnesium hydroxide, hydroxylated Metal hydroxides such as aluminum; anionic surfactants such as sodium lauryl sulfate and calcium stearate; cationic surfactants such as benzalkonium chloride; and polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acids Nonionic surfactants such as esters and sucrose fatty acid esters), stabilizers (paraoxybenzoates such as methylparaben and propylparaben; chlorobutanol, benzyl alcohol, phenylethyl) Alcohols such as alcohol; benzalkonium chloride; phenols, phenols such as cresol; thimerosal; dehydroacetic acid; and sorbic acid and the. ), Flavoring agents (for example, commonly used sweeteners, acidulants, fragrances and the like), and additives such as diluents, and the like.

For the method of introducing the compound of the present invention into a patient, a colloidal dispersion system can be used in addition to the above. The colloidal dispersion system is expected to have an effect of enhancing the stability of the compound in the living body and an effect of efficiently transporting the compound to a specific organ, tissue or cell. Colloidal dispersions are not limited as long as they are commonly used, but are based on lipids including polymer complexes, nanocapsules, microspheres, beads, and oil-in-water emulsifiers, micelles, mixed micelles and liposomes. Dispersed systems can be mentioned, preferably a plurality of liposomes, artificial membrane vesicles that are effective in efficiently transporting compounds to specific organs, tissues or cells (Mannino et al., Biotechniques, 1988). 6,682; Blume and Cevc, Biochem. Et Biophys. Acta, 1990, 1029, 91; Lappalainen et al., Antiviral Res., 1994, 23, 119; Chonn and Cullis, Current Op. Biotech., 1995, 6,698).
Unilamellar liposomes with a size range of 0.2-0.4 μm can encapsulate a significant proportion of aqueous buffer containing macromolecules, and the compounds are encapsulated in this aqueous inner membrane It is transported to brain cells in an active form (Fraley et al., Trends Biochem. Sci., 1981, 6, 77). The composition of liposomes is usually a complex of lipids, especially phospholipids, especially phospholipids with a high phase transition temperature, usually combined with one or more steroids, especially cholesterol. Examples of lipids useful for liposome production include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, sphingolipid, phosphatidylethanolamine, cerebroside and ganglioside. Particularly useful is diacylphosphatidylglycerol, where the lipid moiety contains 14-18 carbon atoms, in particular 16-18 carbon atoms and is saturated (double bonds within the 14-18 carbon atom chain). Is missing). Exemplary phospholipids include phosphatidylcholine, dipalmitoyl phosphatidylcholine and distearoyl phosphatidylcholine.

  Targeting of colloidal dispersions including liposomes can be either passive or active. Passive targeting is achieved by taking advantage of the inherent propensity of liposomes to distribute to the reticuloendothelial cells of organs containing sinusoidal capillaries. On the other hand, active targeting can be achieved by, for example, viral protein coats (Morishita et al., Proc. Natl. Acad. Sci. (USA), 1993, 90, 8474), monoclonal antibodies (or appropriate binding portions thereof). To bind specific ligands such as sugars, glycolipids or proteins (or appropriate oligopeptide fragments thereof) to liposomes, or to achieve distribution to organs and cell types other than naturally occurring localization sites Examples thereof include a method for modifying the liposome by changing the composition of the liposome. The surface of the targeted colloidal dispersion can be modified in various ways. In liposome-targeted delivery systems, lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the target ligand in close association with the lipid bilayer. A variety of linking groups can be used to link the lipid chain to the target ligand. Target ligands that bind to specific cell surface molecules found predominantly on cells where delivery of the oligonucleotides of the invention is desired are, for example, (1) predominantly expressed by the cells where delivery is desired. Polyclonal or monoclonal antibodies that specifically bind to hormones, growth factors or appropriate oligopeptide fragments thereof, or (2) antigenic epitopes predominantly found on target cells, bound to specific cell receptors Or a suitable fragment thereof (e.g., Fab; F (ab ') 2). Two or more bioactive agents (eg, insulin sensitizer-sensitive polypeptide and other agents) can be complexed and administered within a single liposome. Agents that enhance the intracellular stability and / or targeting of the contents can also be added to the colloidal dispersion.

  The amount used varies depending on symptoms, age, etc., but in the case of oral administration, the lower limit is 1 mg (preferably 30 mg), the upper limit is 2000 mg (preferably 1500 mg), and in the case of injection, 1 A lower limit of 0.1 mg (preferably 5 mg) and an upper limit of 1000 mg (preferably 500 mg) can be administered by subcutaneous injection, intramuscular injection or intravenous injection.

EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited to these examples. In the following examples, sugars, insulin, leptin, and DNA were quantified in accordance with the instructions for commercially available products when using commercially available reagents and kits. In addition, the concentration of sugar, total cholesterol, and neutral fat was expressed per deciliter according to the custom.

(Example 1) Difference in the degree of obesity formation in a high-fat diet fed by mouse strain
7-week-old male C57BL / 6N and DBA / 2N mice (purchased from Charles River Japan) and C57BL / KsJ (+ m / + m) mice (purchased from CLEA Japan) high fat diet (Funahashi Farm FR- (2) Lard (ROTTERDAMSCHE MARGARINE INDUSTRIE) added at a rate of 25 g) to 75 g of powdered feed, and then examined obesity formation over 12 weeks (16 animals in each group) . Since the average body weight at the start of the high-fat diet was different (C57BL / 6N: 19.0 g, DBA / 2N: 22.8 g, C57BL / KsJ: 21.6 g), the comparison was made with the weight gain after the start. The results are shown in Figure 1. C57BL / 6N mice showed the most weight gain, gaining weight over 12 weeks, and an average weight gain of 23.4 g at 12 weeks. On the other hand, the weight gain of DBA / 2N mice reached 15.1 g at 8 feeding weeks, but the subsequent weight gain was small, 15.9 g at 12 weeks, compared to C57BL / 6 mice at 12 feeding weeks. A weight difference of 7.5 g was formed. C57BL / KsJ mice showed the slowest weight gain, increasing only 9.6 g in 12 weeks. From the above results, it was clarified that C57BL / 6N mice are easily obese by feeding with a high-fat diet, but DBA / 2N mice and C57BL / KsJ mice are hardly obese.

  DBA / 2N mice were fed a high fat diet until 21 feeding weeks (body weight 41.7 g), but diabetes did not develop.

(Example 2) Development of obesity due to high fat diet in BDF1 mice It is expected to have both C57BL / 6 mice that tend to form obesity due to high fat diet and DBA / 2 mice that are prone to diabetes BDF1 mice were fed a high fat diet to investigate the development of diabetes due to obesity.

  Female C57BL / 6 mice and male DBA / 2 mice F1 hybrid [(C57BL / 6N x DBA / 2N): F1] 7-week-old male BDF1 mice (purchased from Charles River, Japan) Obesity formation and the onset of diabetes were studied over 14 weeks for the group (16 animals) fed the pellets (Funabashi Farm) and the group fed the high fat diet (16 animals).

  The change in average body weight of both groups is shown in FIG. 2A. The body weight of the high-fat diet group increased markedly from the start of the experiment to 8 feeding weeks, and was about 10 g higher than that of the normal diet group. After that, both groups gained weight at approximately the same rate, and the difference in weight between the two groups remained at about 10 g even after 14 weeks (14 feeding weeks). In order to confirm the increase in body fat due to obesity, various tissue weights were measured during 14 feeding weeks (FIG. 2B). In the high fat diet group, mesenteric fat (visceral fat) and groin subcutaneous fat (subcutaneous fat) significantly increased. In addition, the liver weight increased remarkably, and the appearance was white compared with the liver in the normal diet, strongly suggesting the presence of fatty liver.

(Example 3) Diabetes onset by high fat diet rearing with BDF1 mice (1) Rearing mice and obtaining plasma, urine and tissues
The onset of diabetes was examined over 14 weeks in the group fed with a normal diet and a high fat diet to male BDF1 mice at 7 weeks of age as in Example 1. Urine collection and blood collection were performed between 9 am and 10:30 am under free feeding.

  Blood was collected from the tail vein of mice using a heparinized hematocrit tube (manufactured by HIRSCHMANN LABORGERATE), and the blood was centrifuged at 10,000 rpm for 5 minutes to prepare plasma and stored frozen. For urine, spontaneous urine immediately after urination was collected and stored frozen. For tissue weight measurement, the animals were decapitated under ether anesthesia, and various adipose tissues and livers were excised and their wet weights were measured.

(2) Measurement of blood glucose level and urinary glucose level The blood glucose level and urinary glucose level in the plasma of mice were measured by the Murotase / GOD method using glucose CII-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). FIG. 3A shows changes in individual blood glucose levels after the start of the experiment and average blood glucose levels.

  In the high fat diet group, individuals with hyperglycemia of 301 mg / dl appeared from 8 feeding weeks, and blood glucose levels were statistically significantly increased from 8 feeding weeks to 14 feeding weeks compared to the normal diet group did. The blood glucose level of the normal diet group during the 14 feeding weeks was 179 ± 19 mg / dl (mean ± SD), but 13 of 16 cases (81%) in the high fat diet group had a blood glucose level of 250 mg / dl or higher. Some individuals showed marked hyperglycemia around 500 mg / dl. FIG. 3B shows the relationship between individual urine sugar levels and blood glucose levels during the 14 feeding weeks. The urinary glucose level in the normal diet group during the 14 feeding weeks was 13.2 ± 5.0 mg / dl (mean ± SEM), and no individual exceeded 30 mg / dl. The urinary glucose level in the high fat diet group was positively correlated with the blood glucose level, showing a markedly high level of about 10,000 mg / dl at the maximum. 73% of the animals (11/15 cases) measured showed urinary glucose values of 30 mg / dl or higher. In addition, the erythrocyte glycated hemoglobin (HbA1c) rate, which is thought to reflect a persistent hyperglycemic state, was statistically significantly increased in the high-fat diet group at 14 feeding weeks (FIG. 3C).

  The relationship between the body weight and blood glucose level of the high fat diet group is shown in FIG. 3D. Individuals with body weight between 20 g and about 47 g did not significantly change blood glucose levels, but individuals with body weights exceeding about 48-50 g showed a rapid increase in blood glucose levels, Weight was related. This means that there is a simple obesity period with a body weight of 47 g or less without diabetes, and at a weight of about 48-50 g, there is an obesity-dependent diabetic period, both periods being clearly separated. It is present. Human obesity is known to shift from normal glucose tolerance obesity to glucose intolerance and then to diabetes, and the phenotype of obesity / diabetes in high-fat diet-fed BDF1 mice This model is considered to be a model that progresses from obesity to type 2 diabetes, which is similar to the phenotype.

(3) Insulin concentration in plasma The insulin concentration in plasma was measured with an ultrasensitive rat insulin measurement kit (Seikagaku Corporation) using mouse insulin as a standard product. Plasma leptin concentration was measured with a mouse leptin measurement kit (Seikagaku Corporation).

  The plasma insulin concentration increased with time in both the high fat diet group and the normal diet group, but more significantly in the high fat diet group. Plasma leptin concentrations increased with body weight in both groups (Table 1).

(4) Lipid concentration in plasma Total cholesterol concentration in plasma was measured with cholesterol CII-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). In addition, the plasma free fatty acid concentration was measured with NEFA C-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). Plasma triglyceride concentration was measured using GPO-TRINDER (SIGMA).

  After 8 feeding weeks, the plasma total cholesterol level increased in the high fat diet group compared to the normal diet group, but the plasma triglyceride concentration decreased (Table 2). An increase in plasma total cholesterol concentration and a decrease in plasma neutral fat concentration were observed before an increase in blood glucose level was observed. On the other hand, the plasma free fatty acid concentration did not change regardless of the type of food.

(Example 4) Analysis of glucose tolerance and insulin secretion during glucose tolerance test in diabetes in BDF1 mice bred on high fat diet (1) Selection of diabetic mice and non-diabetic mice Breeding on high fat diet for 14-18 weeks Among these mice, mice exhibiting a blood glucose level of 300 mg / dl or higher under feeding conditions were mice that developed diabetes (diabetic mice), and mice below that were non-diabetic mice.

(2) Measurement of glucose tolerance
Glucose tolerance was measured using BDF1 mice at 14-18 feeding weeks. BDF1 mice (normal diet group, 7 mice) fed normal diet and BDF1 mice (high fat diet / diabetes group: average blood glucose level of 440 mg / dl, 7 mice) that developed diabetes on high fat diet The experiment was conducted in three groups of BDF1 mice (high fat diet / non-diabetic group: average blood glucose level of 225 mg / dl, 6 mice under feeding conditions) in which fat symptoms were not observed. After fasting overnight, glucose solution (500 mg / ml) was orally administered at a dose of 2.0 g / kg (4.0 ml / kg), and 0, 10, 15, 30, 45, and 60 minutes after administration Blood was collected from the tail vein. The blood was centrifuged at 10,000 rpm for 5 minutes to separate plasma.

  Plasma glucose concentration was measured using Glucose CII-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). Plasma insulin concentration was measured using an ultrasensitive rat insulin measurement kit (Seikagaku Corporation).

  The blood sugar levels at 30 minutes and 60 minutes after high-fat diet / non-diabetic group and normal group were 373 ± 40 and 408 ± 53 mg / dl (mean ± SEM), 378 ± 33 and 360 ± 42, respectively. No significant difference was observed in mg / dl (mean ± SEM), and glucose tolerance was maintained in the high fat diet / non-diabetic group almost equal to that in the normal diet group. On the other hand, in the high-fat diet / diabetic group, the blood glucose level after oral glucose load markedly increased compared to the normal diet group (the blood glucose levels at 30 minutes and 60 minutes after the glucose load were 610 ± 37 and 678). P value was 0.0012 and 0.0013 at ± 37 mg / dl (mean ± SEM), and abnormal glucose tolerance appeared clearly (FIG. 4A). Plasma insulin levels increased after glucose load in all BDF1 diet groups (FIG. 4B). However, although markedly increased insulin secretion was observed in the high fat diet / non-diabetic group, insulin secretion was clearly decreased in the high fat diet / diabetic group compared to the high fat diet / non-diabetic group. The relationship between blood glucose AUC (Area Under the Curve) and plasma insulin concentration AUC for 60 minutes after oral glucose load shows a negative correlation, and individuals with poor glucose tolerance have insulin. It was revealed that the secretion ability was low (FIG. 4C).

  These results indicate that the increase in blood glucose levels observed in obese / diabetic BDF1 mice was due to a decrease in insulin secretion that had increased to cope with the deterioration of insulin resistance due to obesity. It was thought that it was in the state of.

  The onset of diabetes due to failure of compensatory hypersecretion of insulin observed in this high fat dietary obese BDF1 mouse is very similar to the pathogenesis of obesity to type 2 diabetes in humans. That is, when obesity is formed in humans, insulin resistance is induced and hyperinsulinemia develops compensably. Later, insulin secretion decreases due to exhaustion of insulin-secreting cells on the islets of Langerhans, and it is thought that diabetes develops due to relative insulin deficiency (Cavaghan MK et al., J Clin Invest, 106: 329-333, 2000). ).

  As the mechanism of compensatory insulin secretion failure in the high-fat diet / obese diabetic BDF1 mouse shown this time, it was thought that the decrease in pancreatic β-cell insulin content and the glucose-induced insulin secretion ability were important.

(Example 5) Measurement of insulin content and insulin secretory capacity in diabetes in BDF1 mice reared on a high fat diet (1) Isolation of islets of Langerhans
After sacrifice of BDF1 mice at 14 feeding weeks, pancreas was removed from the common bile duct with 2.5 ml collagenase solution [4 mg / ml type XI collagenase (SIGMA), and HBSS containing 2 mg / ml bovine serum albumin (BSA) ( Hanks' Balanced Salt Solution)] and excised and incubated at 37 ° C. for 3 minutes and 30 seconds. The tissue was suspended in 30 ml of albumin solution (HBSS containing 2 mg / ml BSA), left on ice for 2 minutes, the supernatant was removed, and the islets of Langerhans were isolated and collected under a stereomicroscope.

(2) Measurement of insulin content
Insulin content was measured using the islets of Langerhans collected from BDF1 mice at 14 feeding weeks.

  In Langerhans Island, a high-fat diet / diabetic group, there were many Langerhans islands that were enlarged compared to the normal diet group (Fig. 6). By measuring the Langerhans DNA content and dividing the insulin content, The difference in the number of cells contained in the island was corrected. The average of the islets of Langerhans in the high fat diet / diabetic group was 2.3 ng / ng DNA, which was significantly lower than the average (6.0 ng / ng DNA) in the normal diet group (FIG. 5). At this time, 2 cases that did not develop diabetes even under high fat diet conditions showed insulin content equivalent to that of the normal diet group.

(3) Measurement of insulin secretion
For in vitro insulin secretion test of isolated islets of Langerhans, Krebs-Ringer bicarbonate buffer (KRB buffer: 129 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO ₄ , 1.2 mM KH ₂ PO ₄ , 2.5 mM CaCl ₂ , 5 mM NaHCO ₃ , 10 mM HEPES, pH 7.4, containing 0.2% BSA). 14 Pre-incubation of islets of Langerhans collected from BDF1 during the feeding week with KRB buffer containing 5.6 mM glucose for 30 minutes at 37 ° C, followed by KRB buffer containing 5.6, 11.1 or 22.2 mM glucose and 5.6 mM glucose containing high potassium In KRB buffer (containing 85 mM NaCl, 48.8 mM KCl, 1.2 mM MgSO ₄ , 1.2 mM KH ₂ PO ₄ , 2.5 mM CaCl ₂ , 5 mM NaHCO ₃ , 10 mM HEPES, pH 7.4, 0.2% BSA) Incubated for 60 minutes at 37 ° C., the supernatant was diluted with 0.5% BSA solution, and the insulin concentration was measured using an ultrasensitive rat insulin measurement kit (Seikagaku Corporation).

  The islets of Langerhans used in the experiment were sonicated for 30 seconds (Handy Sonic, output 10), and the DNA concentration was measured using a PicoGreen DNA quantitation kit (Molecular Probe). To eliminate the effect of the difference in the size of the islets of Langerhans, the difference in the number of cells was corrected by dividing the amount of insulin secretion by the DNA content of the islets of Langerhans.

  In in vitro glucose stimulation experiments, the islets of Langerhans collected from the normal diet group activated insulin secretion as the glucose concentration increased, whereas the islets of the high-fat diet / diabetic group responded differently. It was shown (repeated measurement analysis of variance P = 0.025), and until 11.1 mM glucose stimulation, the response was equivalent to that of the normal diet group, but insulin secretion was rather decreased by 22.2 mM glucose stimulation (FIG. 6).

(Example 6) Insulin positive cells in the islets of Langerhans (1) Color reaction
The pancreas excised from a 15-week-feeding mouse was fixed with a Buan solution (saturated picric acid: formalin: acetic acid = 15: 5: 1) at 4 ° C. for 4 hours, decolorized with ethanol, and then paraffin-wrapped according to a conventional method Buried. Serial sections sliced to 3 μm thickness were deparaffinized with 100% xylene (5 minutes, 2 times) and 100% ethanol (5 minutes, 2 times). After drying, blocking was performed with 0.3% hydrogen peroxide solution for 30 minutes, and then with phosphate buffered saline containing 5% BSA for 30 minutes.

  Using rabbit anti-insulin (H-86) antibody (Santa Cruz, SC-9168, dilution ratio 1/300) and rabbit anti-glucagon antibody (Zymed, 18-0064, dilution ratio 1/1000) as primary antibodies, Sections were incubated overnight at 4 ° C. As a secondary antibody, biotinylated anti-rabbit IgG (Vector, BA-1000, dilution ratio 1/200) was used, and serial sections were reacted at room temperature for 1 hour. Then, it was reacted with a horse radish peroxidase labeled avidin-biotin complex (Vector, Vectastain, PK-6100) at room temperature for 45 minutes, and serial sections were developed using AEC (Zymed, 00-2007).

(2) Immunohistochemical analysis of the islets of Langerhans In the normal size of Langerhans islets (diameter of about 150 μm) found in mice in the normal diet group, insulin-positive cells that are strongly stained and scattered around the periphery Glucagon-positive cells were observed, indicating cell localization unique to mouse islets of Langerhans (A and B in FIG. 7). The stained images of normal-sized Langerhans islands in the high-fat diet / non-diabetic group were similar to those in the normal diet group, and many enlarged Langerhans islands were observed, but the localization pattern of insulin-positive cells and glucagon-positive cells was It was the same as in the case of a normal diet (E and F in Fig. 7). On the other hand, in the high-fat diet / diabetes group, β-cells with decreased insulin staining were observed in normal-sized islets of Langerhans and a large number of enlarged islets of Langerhans (G and I in FIG. 7). The localization of insulin-positive β-cells and glucagon-positive α-cells in the islets of Langerhans was clearly different from those in the normal diet group and the high-fat diet / non-diabetic group (H and J in Fig. 7). .

  In insulin resistance states that do not involve impaired glucose tolerance, such as simple obesity in humans, insulin resistance is thought to be compensated by hypertrophy of the islets of Langerhans (proliferation of β cells) (Kloppel G et al., Surv Synth Pathol Res, 4: 110-125, 1985).

  In this experiment, the islets of obese BDF1 mice were significantly enlarged compared to normal diet-fed DBF1 mice, and β-cell proliferation was observed. However, a large number of β-cells with reduced insulin immunoreactivity were found in the enlarged islets of Langerhans in diabetic mice, suggesting the possibility that insulin synthesis function is reduced. The β cells of this model mouse were considered to correspond to the β cell exhaustion state assumed in human type 2 diabetes.

(Example 7) Presence of obesity and diabetes in C57BL / 6 mice reared on high fat diet
We investigated whether the onset of diabetes due to obesity to high fat diet observed in BDF1 mice was also observed in C57BL / 6 mice. Diabetes in BDF1 mice was observed in high fat diets for 12 weeks and obesity over a certain weight (approximately 48 g to 50 g), so 18 diets of high fat diet that met both conditions Experiments were performed using mice with an average body weight of about 50 g per week. The average body weight of the high-fat diet group reached 50.1 g, which was markedly obese, about 15 g heavier than the normal diet group. C57BL / 6 mice are shorter than BDF1 mice (average body length of C57BL / 6 mice is 102 mm and BDF1 mice are 107 mm at 14 weeks of high fat diet) It is considered to be in a more advanced obesity state compared to BDF1 mice.

  The blood glucose level of C57BL / 6 mice raised on a high-fat diet was slightly higher than that of the normal diet group, and no individual showed diabetes (Table 3). The blood insulin level was significantly increased in the high fat diet group, approximately 9 times that in the normal diet group.

OGTT was performed to measure glucose tolerance. Although the blood glucose level after overnight fasting was slightly higher in the high fat diet group, the blood glucose level after glucose loading was not clearly different between the two groups, and the high fat diet obese C57BL / 6 mice were good. Showed high glucose tolerance (FIG. 8A). Insulin secretion after glucose load increased in both groups, but the change was significant in the high-fat diet group, indicating that glucose tolerance was maintained by high insulin secretion ability (FIG. 8B).

The figure which shows the difference by the mouse strain of obesity formation by a high fat diet load. White circles represent C57BL / 6 mice, black squares represent DBA / 2 mice, and gray triangles represent C57BL / KsJ mice. Data are mean ± standard error (n = 16). * P <0.05, ** P <0.001 (comparison of DBA / 2 and C57BL / KsJ to C57BL / 6, respectively). The figure which shows the obesity induced by the high fat diet in a BDF1 mouse | mouth. (A) Changes in body weight when fed on a normal diet (white circle) or a high fat diet (black circle). Each point is an average value ± standard error (n = 16). (B) Adipose tissue [perigonal fat (EPI), mesenteric fat (MES), groin subcutaneous fat (ING) when fed on a normal diet (white column) or a high fat diet (black column) for 14 weeks] , Brown fat (BAT)] and liver weight. Data are mean ± standard error (n = 8). * P <0.05, ** P <0.001 (compared to normal diet). The figure which shows the diabetes induced by a high fat diet in BDF1 mouse | mouth. (A) Blood glucose level was shown by individual (white circle for normal diet, black circle for high fat diet) and by group (dotted line for normal diet, solid line for high fat diet). * P <0.05, ** P <0.001 (Comparison between high fat diet and normal diet). (B) Correlation between the blood glucose level and urine sugar level of each mouse was shown (white circles are normal-fed mice, black circles are high-fat diet-fed mice).

(C) Hemoglobin A1c (HbA1c) concentration was shown. Values are mean ± standard error (n = 16). (D) The correlation between the body weight and blood glucose level of mice raised on a high fat diet was shown. The period of breeding on high fat diet is as follows: white circle is 0 weeks, gray diamond is 8 weeks, black triangle is 12 weeks, asterisk is 14 weeks.
The figure which shows the oral glucose tolerance test in the BDF1 mouse | mouth at the time of 14 feeding weeks. The blood glucose level (A) and blood insulin level (B) after oral administration of glucose were shown. The high fat diet / diabetes (n = 7) is indicated by a black circle, the high fat diet / non-diabetic (n = 6) is indicated by a gray triangle, and the normal diet (n = 7) is indicated by a white circle, and the data represents an average value ± standard error. * P <0.05, ** P <0.001 (high fat diet / diabetes or high fat diet / non-diabetes compared to normal diet), † P <0.05 (high fat diet / non-diabetes compared to high fat diet / diabetes) .

(C) Correlation between AUC of blood glucose level and AUC of blood insulin level in mice fed a high fat diet was shown.
The figure which shows the insulin content in isolated islets of Langerhans. Groups raised on high fat diet were divided into non-diabetic individuals (gray circles) and diabetic individuals (black circles). In addition to the values of each individual, the average value and standard error of the normal diet group and the high fat diet group are also shown (number of cases is 8). * P <0.05 (high fat vs normal). The figure which shows the insulin secretion from the isolated islet of Langerhans at the time of 14 feeding weeks. The normal diet group was represented by a white column, and the high fat diet group was represented by a black column. The concentration of sugar and KCl in the solution in which Langerhans is incubated is shown in the figure. Values are mean ± standard error (values obtained from 6 mice). * P <0.05 (Comparison of high fat diet and normal diet). The figure which shows the immunohistochemical staining of islets of Langerhans. Pancreatic tissues were stained with anti-insulin antibodies (A, C, E, G, I) and anti-glucagon antibodies (B, D, F, H, J). Islets of Langerhans (A and B) in mice fed with normal diet (A and B), or Langerhans islands of mice not fed with high fat diet (high fat diet / non-diabetic) (C, D, E) , F), and Langerhans Island (G, H, I, J) of mice (high fat diet / diabetes) that were bred with a high fat diet and developed diabetes. The scale is 100 mm. The figure which shows the oral glucose tolerance test in a C57BL / 6 mouse. The blood glucose level (A) and insulin level (B) after oral administration of glucose are shown. The white circles represent the normal diet group, the black circles represent the high fat diet group, and the values indicate mean ± standard error (number of cases is 8). * p <0.05, ** p <0.001 (comparison between normal diet group and high fat diet group).

Claims (24)

A method for producing an obese diabetic model mouse comprising the following steps 1) and 2):
1) A mouse selected from C57BL / 6 mouse, 129 / J mouse, MA / J mouse, BALB / c mouse and AKR / J mouse, DBA / 2 mouse, C57BL / KsJ mouse, SWR / J mouse, CBA / Breeding a hybrid mouse with a mouse selected from LT mouse, C3HeB / FeJ mouse, ICR (CD1) mouse and FVB / NJ mouse on a high fat diet;
2) A step of measuring the body weight of the mouse of 1) and selecting an obese mouse. A method for producing an obese diabetic model mouse comprising the following steps 1) and 2):
1) A mouse selected from C57BL / 6 mouse, 129 / J mouse, MA / J mouse, BALB / c mouse and AKR / J mouse, DBA / 2 mouse, C57BL / KsJ mouse, SWR / J mouse, CBA / Breeding a hybrid mouse with a mouse selected from LT mouse, C3HeB / FeJ mouse, ICR (CD1) mouse and FVB / NJ mouse on a high fat diet;
2) A step of measuring the weight of the mouse of 1) and selecting a mouse whose weight has increased by 20% or more compared to a mouse raised on a normal diet. A method for producing an obese diabetic model mouse comprising the following steps 1) and 2):
1) A mouse selected from C57BL / 6 mouse, 129 / J mouse, MA / J mouse, BALB / c mouse and AKR / J mouse, DBA / 2 mouse, C57BL / KsJ mouse, SWR / J mouse, CBA / Breeding a hybrid mouse with a mouse selected from LT mouse, C3HeB / FeJ mouse, ICR (CD1) mouse and FVB / NJ mouse on a high fat diet;
2) A step of examining the blood glucose level and / or urine sugar level of the mouse of 1), and selecting a mouse in which an increase in blood glucose level and / or diabetes has been confirmed as compared with a mouse raised on a normal diet. A method for producing an obese diabetes model mouse comprising the following steps 1) to 3):
1) A mouse selected from C57BL / 6 mouse, 129 / J mouse, MA / J mouse, BALB / c mouse and AKR / J mouse, DBA / 2 mouse, C57BL / KsJ mouse, SWR / J mouse, CBA / Breeding a hybrid mouse with a mouse selected from LT mouse, C3HeB / FeJ mouse, ICR (CD1) mouse and FVB / NJ mouse on a high fat diet;
2) A step of measuring the weight of the mouse of 1) and selecting a mouse whose weight has increased by 20% or more compared to a mouse raised on a normal diet;
3) A step of selecting a mouse in which an increase in blood glucose level and / or diabetes has been confirmed as compared with a mouse raised on a normal diet. A method for producing an obese diabetic model mouse comprising the following steps 1) and 2):
1) A mouse selected from C57BL / 6 mouse, 129 / J mouse, MA / J mouse, BALB / c mouse and AKR / J mouse, DBA / 2 mouse, C57BL / KsJ mouse, SWR / J mouse, CBA / A step of administering and breeding goldthioglucose to a hybrid mouse with a mouse selected from LT mouse, C3HeB / FeJ mouse, ICR (CD1) mouse and FVB / NJ mouse;
2) A step of measuring the body weight of the mouse of 1) and selecting an obese mouse. A method for producing an obese diabetic model mouse comprising the following steps 1) and 2):
1) A mouse selected from C57BL / 6 mouse, 129 / J mouse, MA / J mouse, BALB / c mouse and AKR / J mouse, DBA / 2 mouse, C57BL / KsJ mouse, SWR / J mouse, CBA / Administering a gold thioglucose to a hybrid mouse with a mouse selected from LT mouse, C3HeB / FeJ mouse, ICR (CD1) mouse and FVB / NJ mouse;
2) A step of measuring the body weight of the mouse of 1) and selecting a mouse whose body weight has increased by 20% or more compared to a mouse to which goldthioglucose was not administered. A method for producing an obese diabetic model mouse comprising the following steps 1) and 2):
1) A mouse selected from C57BL / 6 mouse, 129 / J mouse, MA / J mouse, BALB / c mouse and AKR / J mouse, DBA / 2 mouse, C57BL / KsJ mouse, SWR / J mouse, CBA / Administering a gold thioglucose to a hybrid mouse with a mouse selected from LT mouse, C3HeB / FeJ mouse, ICR (CD1) mouse and FVB / NJ mouse;
2) A step of examining the blood glucose level and / or urine sugar level of the mouse of 1) and selecting a mouse in which an increase in blood glucose level and / or diabetes has been confirmed compared to a mouse to which goldthioglucose was not administered. A method for producing an obese diabetes model mouse comprising the following steps 1) to 3):
1) A mouse selected from C57BL / 6 mouse, 129 / J mouse, MA / J mouse, BALB / c mouse and AKR / J mouse, DBA / 2 mouse, C57BL / KsJ mouse, SWR / J mouse, CBA / Administering a gold thioglucose to a hybrid mouse with a mouse selected from LT mouse, C3HeB / FeJ mouse, ICR (CD1) mouse and FVB / NJ mouse;
2) A step of measuring the body weight of the mouse of 1) and selecting a mouse having a body weight increased by 20% or more compared to a mouse not administered with goldthioglucose;
3) A step of selecting a mouse in which an increase in blood glucose level and / or diabetes has been confirmed compared to a mouse to which goldthioglucose has not been administered. The hybrid mouse is an F1 hybrid of C57BL / 6 and DBA / 2 mice [(C57BL / 6 x DBA / 2): F1], any one of claims 1 to 8, characterized in that A method for producing a model mouse for obesity diabetes described in 1. An obese diabetes model mouse produced by the production method according to any one of claims 1 to 9. A method for testing a prophylactic and / or ameliorating substance for at least one disease selected from abnormal glucose metabolism, obesity and abnormal lipid metabolism, comprising the following steps 1) and 2):
1) A step of administering a test substance to the obese diabetes model mouse according to claim 10;
2) Body weight, blood glucose level, urinary glucose level, blood insulin level, blood glucose level and insulin level in glucose tolerance test, blood triglyceride concentration, insulin content or insulin secretory capacity of islets of Langerhans and insulin Measuring at least one selected from reactivity. A method for testing a prophylactic and / or ameliorating substance for at least one disease selected from abnormal glucose metabolism, obesity and abnormal lipid metabolism, comprising the following steps 1) and 2):
1) A step of bringing a test substance into contact with a tissue derived from the obese diabetes model mouse according to claim 10;
2) A step of measuring at least one selected from triglyceride content, insulin content, insulin secretion ability, and insulin responsiveness in the tissue of 1). A method for testing a prophylactic and / or ameliorating substance for at least one disease selected from abnormal glucose metabolism, obesity and abnormal lipid metabolism, comprising the following steps 1) and 2):
1) A step of administering a test substance to the obese diabetes model mouse according to claim 10;
2) A step of measuring at least one selected from triglyceride content, glucose uptake ability and glucose synthesis ability in the tissue extracted from the mouse of 1). A method for testing a prophylactic and / or ameliorating substance for at least one disease selected from abnormal glucose metabolism, obesity and abnormal lipid metabolism, comprising the following steps 1) and 2):
1) A step of administering a test substance to the obese diabetes model mouse according to claim 10;
2) A step of measuring at least one selected from triglyceride content, glucose uptake ability and glucose synthesis ability in the tissue removed from the mouse of 1);
3) A step of selecting a test substance in which at least one selected from a decrease in triglyceride content, an increase in insulin content, an increase in insulin secretion ability, and an increase in insulin reactivity is observed. A method for testing a prophylactic and / or ameliorating substance for at least one disease selected from abnormal glucose metabolism, obesity and abnormal lipid metabolism, comprising the following steps 1) to 3):
1) A step of administering a test substance to the obese diabetes model mouse according to claim 10;
2) Body weight, blood glucose level, urine sugar level, blood insulin level, blood glucose level and insulin level in glucose tolerance test, blood triglyceride concentration, insulin content of Langerhans Island, Langerhans Island Measuring at least one selected from insulin secretory ability and insulin responsiveness;
3) Decrease in body weight, decrease in blood glucose level, decrease in urine sugar level, increase in blood insulin concentration, decrease in blood glucose level or insulin level in glucose tolerance test, decrease in blood triglyceride concentration, Langerhans Island Selecting a test substance in which at least any one selected from an increase in insulin content, an increase in insulin secretory capacity on the islets of Langerhans, and an increase in insulin reactivity is observed. A method for testing a prophylactic and / or ameliorating substance for at least one disease selected from abnormal glucose metabolism, obesity and abnormal lipid metabolism, comprising the following steps 1) to 3):
1) A step of bringing a test substance into contact with a tissue derived from the obese diabetes model mouse according to claim 10;
2) a step of measuring at least one selected from triglyceride content, insulin content, insulin secretion ability and insulin reactivity in the tissue of 1);
3) A step of selecting a test substance in which at least one selected from a decrease in triglyceride content, an increase in insulin content, an increase in insulin secretion ability, and an increase in insulin reactivity is observed. A method for testing a prophylactic and / or ameliorating substance for at least one disease selected from abnormal glucose metabolism, obesity and abnormal lipid metabolism, comprising the following steps 1) to 2):
1) A mouse selected from C57BL / 6 mouse, 129 / J mouse, MA / J mouse, BALB / c mouse and AKR / J mouse, DBA / 2 mouse, C57BL / KsJ mouse, SWR / J mouse, CBA / Administering a test substance to a hybrid mouse with a mouse selected from an LT mouse, a C3HeB / FeJ mouse, an ICR (CD1) mouse and an FVB / NJ mouse, and rearing them on a high fat diet;
2) Body weight, blood glucose level, urinary glucose level, blood insulin level, blood glucose level and insulin level in glucose tolerance test, blood triglyceride concentration, insulin content or insulin secretory capacity of islets of Langerhans and insulin Measuring at least one selected from reactivity. A method for testing a prophylactic and / or ameliorating substance for at least one disease selected from abnormal glucose metabolism, obesity and abnormal lipid metabolism, comprising the following steps 1) to 3):
1) A mouse selected from C57BL / 6 mouse, 129 / J mouse, MA / J mouse, BALB / c mouse and AKR / J mouse, DBA / 2 mouse, C57BL / KsJ mouse, SWR / J mouse, CBA / Administering a test substance to a hybrid mouse with a mouse selected from an LT mouse, a C3HeB / FeJ mouse, an ICR (CD1) mouse and an FVB / NJ mouse, and rearing them on a high fat diet;
2) Body weight, blood glucose level, urine glucose level, blood insulin level, blood glucose level and insulin level in glucose tolerance test, blood triglyceride concentration, insulin content of Langerhans island, insulin secretion of Langerhans island Measuring at least one selected from potency and insulin responsiveness;
3) Decrease in body weight, decrease in blood glucose level, decrease in urine sugar level, increase in blood insulin concentration, decrease in blood glucose level or insulin level in glucose tolerance test, decrease in blood triglyceride concentration, Langerhans Island Selecting a test substance in which at least any one selected from an increase in insulin content, an increase in insulin secretory capacity on the islets of Langerhans, and an increase in insulin reactivity is observed. A method for testing a prophylactic and / or ameliorating substance for at least one disease selected from abnormal glucose metabolism, obesity and abnormal lipid metabolism, comprising the following steps 1) to 3):
1) A step of administering a test substance to a mouse of F1 hybrid [(C57BL / 6 x DBA / 2): F1] of a C57BL / 6 mouse and a DBA / 2 mouse and rearing them on a high fat diet;
2) Body weight, blood glucose level, urine glucose level, blood insulin level, blood glucose level and insulin level in glucose tolerance test, blood triglyceride concentration, insulin content of Langerhans island, insulin secretion of Langerhans island Measuring at least one selected from potency and insulin responsiveness;
3) Decrease in body weight, decrease in blood glucose level, decrease in urine sugar level, increase in blood insulin concentration, decrease in blood glucose level or insulin level in glucose tolerance test, decrease in blood triglyceride concentration, Langerhans Island Selecting a test substance in which at least any one selected from an increase in insulin content, an increase in insulin secretory capacity on the islets of Langerhans, and an increase in insulin reactivity is observed. A method for testing a prophylactic and / or ameliorating substance for at least one disease selected from abnormal glucose metabolism, obesity and abnormal lipid metabolism, comprising the following steps 1) to 2):
1) A mouse selected from C57BL / 6 mouse, 129 / J mouse, MA / J mouse, BALB / c mouse and AKR / J mouse, DBA / 2 mouse, C57BL / KsJ mouse, SWR / J mouse, CBA / Administering a gold thioglucose and a test substance to a hybrid mouse with a mouse selected from an LT mouse, a C3HeB / FeJ mouse, an ICR (CD1) mouse and an FVB / NJ mouse;
2) Body weight, blood glucose level, urinary glucose level, blood insulin level, blood glucose level and insulin level in glucose tolerance test, blood triglyceride concentration, insulin content or insulin secretory capacity of islets of Langerhans and insulin Measuring at least one selected from reactivity. A method for testing a prophylactic and / or ameliorating substance for at least one disease selected from abnormal glucose metabolism, obesity and abnormal lipid metabolism, comprising the following steps 1) to 3):
1) A mouse selected from C57BL / 6 mouse, 129 / J mouse, MA / J mouse, BALB / c mouse and AKR / J mouse, DBA / 2 mouse, C57BL / KsJ mouse, SWR / J mouse, CBA / Administering a gold thioglucose and a test substance to a hybrid mouse with a mouse selected from an LT mouse, a C3HeB / FeJ mouse, an ICR (CD1) mouse and an FVB / NJ mouse;
2) Body weight, blood glucose level, urine glucose level, blood insulin level, blood glucose level and insulin level in glucose tolerance test, blood triglyceride concentration, insulin content of Langerhans island, insulin secretion of Langerhans island Measuring at least one selected from potency and insulin responsiveness;
3) Decrease in body weight, decrease in blood glucose level, decrease in urine sugar level, increase in blood insulin concentration, decrease in blood glucose level or insulin level in glucose tolerance test, decrease in blood triglyceride concentration, Langerhans Island Selecting a test substance in which at least any one selected from an increase in insulin content, an increase in insulin secretory capacity on the islets of Langerhans, and an increase in insulin reactivity is observed. The hybrid mouse is an F1 hybrid of C57BL / 6 and DBA / 2 mice [(C57BL / 6 x DBA / 2): F1], any one selected from claims 17 to 21 4. A test method for a prophylactic and / or ameliorating substance for at least one disease selected from the abnormalities of glucose metabolism, obesity and lipid metabolism. 23. A preventive substance and / or an ameliorating substance for at least one disease selected from among abnormal sugar metabolism, obesity and abnormal lipid metabolism obtained by the method according to claim 11 to 22. A therapeutic agent for at least one disease selected from diabetes, obesity, and hyperlipidemia, comprising the substance according to claim 23 as an active ingredient.