Method for producing non-obese non-human mammalian animal with hyperglycemia

Abstract

To provide a method for manufacturing a non-human mammal with non-obesity type hyperglycemia, which is born with a low birth weight and then develops diabetes without obesity in a normal food environment, as an animal model for diabetes.SOLUTION: The method of manufacturing a non-human mammal with non-obesity type hyperglycemia, in which the blood flow to the uterine artery of a pregnant non-human mammal is cut off so that an embryo is rendered ischemic, includes: a process for rendering the embryo ischemic in the late stage of pregnancy of the pregnant non-human mammal; a process for obtaining a baby non-human mammal which is the embryo that is born; and a process for breeding the baby non-human mammal on a regular diet.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing a non-obese non-human mammalian animal suffering from hyperglycemia. [Background technology]

[0002] As animal models of diabetes, BB rats, LEW IAR1 / iddm rats, Nonobase Diabetic mice, and Akita mice have been reported as genetic models of type 1 diabetes. ZDF rats and Goto-Kakizaki rats have been reported as genetic models of type 2 diabetes. In addition, chemically induced diabetes models have been reported using streptozotocin and Allozan, which are chemicals that induce diabetes (Non-Patent Document 1).

[0003] In animal models of type 1 diabetes characterized by absolute insulin deficiency, the pathways that lead to type 1 diabetes are limited, raising doubts about their relevance to human type 1 diabetes. On the other hand, in animal models of type 2 diabetes characterized by insulin resistance, most of them have abnormalities in obesity-related genes, which leads to insulin resistance and impaired glucose tolerance.

[0004] In recent years, it has become known that low birth weight infants are at high risk of developing obesity and lifestyle-related diseases in adulthood. Fetuses exposed to malnutrition develop a constitution adapted to malnutrition due to changes in the genomic gene expression pattern. This constitution is maintained even after birth, and the infant grows up with a constitution adapted to malnutrition. For such infants, the normal nutritional supply after birth is relatively excessive, and the excess energy is stored as body fat, increasing the risk of childhood obesity and inheriting obesity in adulthood. The idea of ​​fetal programming, which states that changes in constitution during the uterine period are the cause of lifestyle-related diseases after birth, is accepted as a concept called the Developmental Origin of Health and Disease (DOHaD) hypothesis. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Amin Al-Awar et al., Experimental Diabetes Mellitus in Different Animal Models. J Diabetes Res. 2016;2016:9051426. Summary of the Invention [Problem to be solved by the invention]

[0006] There are animal models in which low birth weight infants develop diabetes accompanied by obesity in a nutritious dietary environment after birth. However, low birth weight infants have a high risk of developing diabetes without obesity in a normal dietary environment. For research into the risk of diabetes in low birth weight infants, an animal model in which low birth weight infants develop diabetes without obesity in a normal dietary environment after birth would be useful, but no such animal model exists.

[0007] Therefore, an object of the present invention is to provide a method for producing a non-obese non-human mammalian animal suffering from hyperglycemia. [Means for solving the problem]

[0008] The present invention includes the following aspects. [1] A method for producing a non-obese hyperglycemic non-human mammal, comprising the steps of: putting a fetus of a pregnant non-human mammal into an ischemic state during the late stages of pregnancy; and obtaining a non-human mammal offspring from the fetus that has now been born. [2] The method for producing a non-obese hyperglycemic non-human mammal described in [1], further comprising a step of raising the baby non-human mammal on a normal diet. [3] A method for producing a non-obese hyperglycemic non-human mammal described in [1] or [2], comprising blocking blood flow in the uterine artery of the pregnant non-human mammal to put the fetus into an ischemic state. [4] The method for producing a non-obese hyperglycemic non-human mammal according to any one of [1] to [3], wherein the offspring non-human mammal is a female. Effect of the Invention

[0009] According to the present invention, there is provided a method for producing a non-obese non-human mammal suffering from hyperglycemia. [Brief description of the drawings]

[0010] [Figure 1] An example of blocking the blood flow of a mouse uterine artery with a clip is shown. [Diagram 2] Birth weights of ischemic and non-ischemic groups are shown. [Diagram 3] The graph shows changes in body weight when fed a normal diet or a high-fat diet. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] In one aspect, the present invention provides a method for producing a non-obese hyperglycemic non-human mammal, the method of this aspect comprising the steps of: putting a fetus of a pregnant non-human mammal into an ischemic state during the late stage of pregnancy (hereinafter also referred to as "step (i)"); and obtaining a baby non-human mammal from the fetus (hereinafter also referred to as "step (ii)").

[0012] According to the method of this embodiment, a non-obese non-human mammalian animal can be produced. "Non-obese non-human mammalian animal with hyperglycemia" means a non-human mammalian animal that can develop non-obese hyperglycemia. "Non-obese hyperglycemia" means hyperglycemia not accompanied by obesity. A non-obese non-human mammalian animal with hyperglycemia develops non-obese hyperglycemia when kept under specific conditions.

[0013] "Obesity" refers to a state of being overweight compared to the average body weight (hereinafter also referred to as "normal body weight") of normal individuals of the same species when fed a normal diet. For example, an animal can be said to be obese when its body weight is 1.1 times or more, 1.2 times or more, or 1.3 times or more of its normal body weight. Also, an animal can be said to be non-obese when its body weight is 1.2 times or less, less than 1.2 times, less than 1.1 times, or less than 1.1 times its normal body weight.

[0014] "Hyperglycemia" refers to a state in which blood glucose level is higher than the average blood glucose level (hereinafter also referred to as "normal blood glucose level") of normal individuals of the same species when fed a normal diet. For example, blood glucose level that is 1.3 times or more, 1.4 times or more, 1.5 times or more, 1.6 times or more, 1.7 times or more, or 1.8 times or more of the normal blood glucose level can be said to be hyperglycemia.

[0015] The non-human mammal is not particularly limited. Examples of the non-human mammal include, but are not limited to, non-human primates (monkeys, chimpanzees, gorillas, common marmosets, cynomolgus monkeys, etc.), rodents (mouse, rats, guinea pigs, hamsters, etc.), dogs, cats, rabbits, cows, pigs, horses, goats, sheep, etc. Among these, rodents are preferred, and mice are more preferred, because they are easy to obtain and test.

[0016] The genetic background of the non-human mammal is not particularly limited, but preferably does not have the genetic background of type 1 diabetes or type 2 diabetes. That is, it is preferable that the non-human mammal does not have the causative gene of type 1 diabetes that a type 1 diabetes model animal has and the causative gene of type 2 diabetes that a type 2 diabetes model animal has. When the non-human animal is a mouse, for example, an ICR strain, a C57BL / 6 strain, a BALB / c strain, a DBA / 2 strain, a C3H strain, etc. can be used, but is not limited to these.

[0017] [Step of putting the fetus into an ischemic state: step (i)] In step (i), the fetus of a pregnant non-human mammal is subjected to an ischemic state during the late stages of gestation.

[0018] A pregnant non-human mammal can be obtained by a known method. For example, a male and a female in the breeding season may be housed together and allowed to mate naturally, thereby making the female pregnant. For example, one male may be housed with one or more females (e.g., two or three females). The non-human mammal can be bred according to a method known to those skilled in the art. Pregnancy can be determined by a known method depending on the type of non-human mammal. For example, in the case of mice, females that have mated become pregnant with a high probability, so the day on which a vaginal plug is confirmed can be considered as day 0 of pregnancy. Additionally, a pregnant non-human mammal may be obtained by artificial insemination, or an already pregnant female may be purchased. The pregnant non-human mammal can be reared by a known method depending on the type of the non-human mammal.

[0019] "Late pregnancy" refers to the period from 2 / 3 of the pregnancy to birth. "Pregnancy" refers to the period from mating or implantation of a fertilized egg to birth. The fetus is preferably placed in an ischemic state after 3 / 4, 4 / 5, 5 / 6, or 6 / 7 of the pregnancy. The fetus is preferably placed in an ischemic state before 11 / 12, 10 / 11, 9 / 10, or 8 / 9 of the pregnancy. The fetus can be placed in an ischemic state, for example, after 3 / 4 and before 11 / 12 of the pregnancy. Preferably, the fetus can be placed in an ischemic state after 4 / 5 and before 10 / 11 of the pregnancy, and more preferably, after 5 / 6 and before 9 / 10 of the pregnancy. When the non-human mammal is a mouse, the gestation period is 18 to 20 days (usually 19 days), and the mouse can be put into an ischemic state on days 16 to 17 of gestation, for example.

[0020] The method of putting the fetus in an ischemic state is not particularly limited. For example, the method of putting the fetus in an ischemic state includes a method of blocking the blood flow of the uterine artery of a pregnant non-human animal (hereinafter also referred to as "mother animal"). For example, under appropriate anesthesia conditions, the abdomen of the mother animal is incised to expose the uterine artery, and the blood flow of the uterine artery is blocked using a clip or the like (see FIG. 1). This prevents blood flow to the fetus, and the fetus can be put into an ischemic state. The mother animal can be anesthetized by a known method such as isoflurane inhalation. While the blood flow of the uterine artery is blocked, the body of the mother animal is preferably kept warm at a temperature about body temperature. The death of the mother animal and the fetus can be prevented by keeping the body warm. When the non-human animal is a small animal such as a mouse or a rat, the warming can be performed using, for example, a hot plate.

[0021] The time for putting the fetus into an ischemic state (hereinafter also referred to as "ischemic time") is not limited, but can be a time that does not cause the death of the fetus. When there are multiple fetuses in the uterus, the ischemic time may be a time that allows at least one fetus to survive. The ischemic time can be appropriately selected depending on the type of non-human animal. The ischemic time can be, for example, 30 minutes or less, 25 minutes or less, 20 minutes or less, 19 minutes or less, 18 minutes or less, 16 minutes or less, or 15 minutes or less. The lower limit of the ischemic time is not particularly limited, but can be, for example, 1 minute or more, 3 minutes or more, 5 minutes or more, 7 minutes or more, 10 minutes or more, 12 minutes or more, 13 minutes or more, 14 minutes or more, or 15 minutes or more. The ischemic time can be, for example, 1 to 30 minutes, and preferably 3 to 20 minutes, 5 to 18 minutes, 10 to 17 minutes, or 12 to 15 minutes. When the non-human mammal is a mouse, the ischemic time can be, for example, 15 minutes. When a fetus is put into an ischemic state by obstructing blood flow in the uterine artery with a clip or the like, the time during which blood flow in the uterine artery is obstructed can be regarded as the ischemic time.

[0022] After a predetermined ischemic time has elapsed, the ischemic state of the fetus is released. If the fetus is put into an ischemic state by blocking the blood flow of the uterine artery with a clip or the like, the clip or the like is removed after the predetermined ischemic time has elapsed to restore the blood flow of the uterine artery. The fetus is then returned to the abdominal cavity of the mother animal, and the abdomen of the mother animal is sutured. The mother animal is then kept until delivery.

[0023] [Step (ii) of obtaining a non-human mammalian offspring] In step (ii), a non-human mammalian offspring resulting from the fetus being born is obtained.

[0024] After the step (i), the mother animal is continued to be raised and allowed to give birth. The birth may be natural birth or cesarean section. In this way, a non-human mammalian baby can be obtained from the fetus that has been put into an ischemic state in the step (i) and has been brought to birth. In this step, it is preferable to obtain female non-human mammals from the offspring, as females tend to be more susceptible to developing non-obese hyperglycemia.

[0025] The baby non-human mammal obtained in this step has a low birth weight compared to a baby non-human mammal born from a mother animal whose pregnancy was allowed to continue normally without carrying out step (i). This baby non-human mammal can be made to develop hyperglycemia without obesity by feeding it a normal diet, as described below. Therefore, it can be used as a model animal for the development of non-obese hyperglycemia.

[0026] [Optional process] The method of this embodiment may include an optional step in addition to the above steps (i) and (ii). An example of the optional step is a step of feeding the baby non-human mammal with a normal diet (hereinafter, also referred to as "step (iii)").

[0027] <Step (iii) of feeding the rats with normal food> By raising the baby non-human mammal obtained in the above step (ii) on a normal diet, it is possible to cause the baby to develop non-obese hyperglycemia. "Normal diet" refers to a diet used in normal rearing of non-human mammals. Normal diet may contain a balanced amount of calories, neither too much nor too little, to maintain the growth of the non-human mammal. Normal diet may be a standard feed for the non-human mammal. For example, when the non-human mammal is a mouse, rat, or hamster, the normal diet may be standard feed for mice, rats, and hamsters MF (Oriental Yeast Co., Ltd.).

[0028] The rearing with a normal diet can be carried out after the baby non-human mammal is weaned. The weaning time of the baby non-human mammal is known depending on the type of the non-human mammal. For example, when the non-human mammal is a mouse, it can be weaned at 4 weeks of age. The rearing with a normal diet can be carried out by a known method depending on the type of the non-human mammal.

[0029] After weaning, when a baby non-human mammal is raised on a normal diet, the body weight usually remains at a level lower than the normal weight and does not become obese. Meanwhile, the fasting blood glucose level rises above the normal blood glucose level. In other words, non-obese hyperglycemia develops. Furthermore, non-human mammals that have developed non-obese hyperglycemia tend to have a higher body fat percentage than the norm. This is presumably because the ischemic state during the fetal period is similar to a state of undernutrition, and the non-obese hyperglycemia mouse has a constitution adapted to the state of undernutrition. Therefore, the non-obese hyperglycemic mouse obtained by the method of this embodiment can be used as a non-obese hyperglycemic mouse that proves the fetal programming hypothesis (DOHaD).

[0030] The baby non-human mammal obtained in step (ii) may be fed with any diet, not just a normal diet. The baby non-human mammal can be a model animal for low birth weight infants, and it is considered that the reaction of low birth weight infants to any diet can be simulated. For example, the baby non-human mammal may be fed with a high-fat diet instead of a normal diet. The term "high-fat diet" refers to a diet with a higher fat content than a normal diet. The high-fat diet may be, for example, a high-fat sample containing 40% or more, 45% or more, 50% or more, 55% or more, or 60% or more of fat in terms of calorie ratio. Examples of the high-fat sample include a high-fat sample HFD-60 (Oriental Yeast Co., Ltd.) with a calorie ratio of 60% fat.

[0031] According to the method of this embodiment, a non-obese non-human mammalian animal can be obtained that develops hyperglycemia. The non-obese non-human mammalian animal that develops hyperglycemia obtained by the method of this embodiment is born with low body weight and can develop hyperglycemia without obesity, and can therefore be used as a model animal for proving the DOHaD hypothesis. In addition, the non-obese non-human mammalian animal can be used in research on the risk of diabetes without obesity in low birth weight infants, treatment and health intervention research for diabetes patients without obesity, research on the development of a marker for predicting the onset of diabetes patients without obesity, treatment and health intervention research for preventing the onset of diabetes patients without obesity, and the like. EXAMPLES

[0032] The present invention will be described below with reference to experimental examples, but the present invention is not limited to the following experimental examples.

[0033] [Creation of non-obese hyperglycemic mice] On day 16.5 of pregnancy, pregnant ICR mice were anesthetized with isoflurane inhalation (induction 5%, maintenance 2%) and had their lower abdomen incised. The uterine artery was exposed, and the blood flow of the uterine artery was blocked with a clip to avoid damaging the fetal mouse (see Figure 1). The mice were left ischemic for 15 minutes. During the ischemic period, the mother mouse was warmed to 37.5°C on a hot plate. The clip on the uterine artery was then removed, the fetus was returned to the abdominal cavity of the mother mouse, and the lower abdomen was sutured. On day 16.5 of pregnancy, the same anesthesia and lower abdominal incision were performed as in the ischemic group, and the non-ischemic control group was created by not blocking the uterine artery. Pregnancy was continued in both the ischemic and non-ischemic groups, and newborn mice were born on day 19 of pregnancy.

[0034] [Raising young mice] The pups were weaned at 4 weeks of age. In each of the ischemic and non-ischemic groups, the pups were divided into two groups: one was a normal diet group and the other was a high-fat diet group. The normal diet group was fed a normal diet (standard diet for mice, rats, and hamsters MF, Oriental Yeast Co., Ltd.) ad libitum from the age of 5 weeks. The high-fat diet group was fed a high-fat diet (high-fat diet with 60% calorie content, HFD-60, Oriental Yeast Co., Ltd.) ad libitum from the age of 5 weeks.

[0035] After birth, pups were weighed twice weekly. At 7 weeks of age, urine was collected using a metabolic gauge to measure β2-microglobulin (β2), microalbumin (Alb), and creatine (Cr). At 8 weeks of age, the mice were fasted for 12 hours, and then anesthetized with isoflurane by inhalation. Body weight and body composition were measured. Blood was collected by cardiac puncture, and fasting blood glucose was measured.

[0036] [result] (birth weight) Figure 2 shows the birth weights of the ischemic and non-ischemic groups. The birth weights of the ischemic group were lower than those of the non-ischemic group. This result indicates that low birth weight pups can be obtained by subjecting the fetus to an ischemic state during the late stages of pregnancy.

[0037] (Weight transition) FIG. 3 shows the changes in body weight of females in the normal diet group and the high-fat diet group. When fed a normal diet, the ischemic group (females) grew at a lower body weight than the non-ischemic group (females), whereas when fed a high-fat diet, the ischemic group (females) developed significant obesity.

[0038] (Blood Glucose Levels) Table 1 shows the body weight and blood glucose level measured after 12 hours fasting at 8 weeks of age. In the ischemic group, blood glucose level was significantly higher than in the non-ischemic group in both normal and high-fat diets. In the ischemic group, blood glucose level was higher in both normal and high-fat diets.

[0039] [Table 1]

[0040] (Body Composition) Table 2 shows the body composition measured at 8 weeks of age after a 12-hour fast. In the ischemic group, FM was larger and FFM was lower than in the non-ischemic group, both in the normal and high-fat diets. On the other hand, there was no difference in BMI between the ischemic and non-ischemic groups in the normal diet. In the high-fat diet, BMI was larger in the ischemic group than in the non-ischemic group.

[0041] [Table 2]

[0042] The meanings of the abbreviations in Table 2 are as follows: TBW: Total Body Water ECF: Extra-Cellular Fluid ICF: Intra-Cellular Fluid FM: Fat Mass FFM: Fat Free Mass BMI: BMI index (Body Mass Index)

[0043] (Urine test) Table 3 shows the results of a urine test performed at 7 weeks of age. Urinary β2 microglobulin (β2) was higher in the ischemic group than in the non-ischemic group when fed a normal diet. Urinary microalbumin (Alb) tended to be higher in the ischemic group than in the non-ischemic group when fed a normal diet and a high-fat diet. Urinary creatine (Cr) was higher in the ischemic group than in the non-ischemic group when fed a normal diet.

[0044] [Table 3] [Industrial Applicability]

[0045] According to the present invention, there is provided a method for producing a non-obese non-human mammal suffering from hyperglycemia.

Claims

1. subjecting a pregnant non-human mammal to an ischemic condition during late pregnancy; Obtaining a non-human mammalian offspring resulting from the fetus being born; and measuring the fasting blood glucose of said offspring non-human mammal The present invention relates to a method for producing a non-obese non-human mammalian animal suffering from hyperglycemia, the method comprising the steps of:

2. Feeding the baby non-human mammal on a normal diet; The method for producing a non-obese non-human mammal suffering from hyperglycemia according to claim 1, further comprising:

3. 3. The method for producing a non-obese hyperglycemic non-human mammal according to claim 1 or 2, wherein the fetus is put into an ischemic state by blocking blood flow in a uterine artery of the pregnant non-human mammal.

4. The method for producing a non-obese non-human mammalian animal suffering from hyperglycemia according to any one of claims 1 to 3, wherein the offspring non-human mammalian animal is a female.

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