Animal Experiments - Rat Diabetes Model

Diabetes mellitus (DM) has become a serious public health problem that endangers human health. DM and its complications not only seriously affect the quality of life of diabetic patients, but also an important cause of disability and death. Therefore, it is particularly important to establish a suitable animal model of diabetes and elucidate the pathogenesis of DM and its complications. At present, the preparation methods of DM animal models mainly include: surgical excision of pancreas, chemical drug induction, spontaneous diabetes animal models, transgenic animals, etc.

First, the DM model of excision of pancreas

Dogs, cats, and rats are often molded, and the pancreas of the experimental animals is removed in whole or most, but the pancreatic duodenal artery anastomosis arch is preserved. If the blood glucose value exceeds 11.1 mmol/L for two consecutive days or the glucose tolerance test is not restored to the pre-injection level for two consecutive days, dm molding is considered successful. The mechanism is that after all or most of the pancreas is removed, permanent DM is produced β cell deletion.

Second, chemical drug-induced DM model

DM can be induced by intraperitoneal injection of streptozotocin or tetraoxypyrimidine, and commonly used animals include mice, rats, rabbits, and dogs. The reference dose of streptozotocin STZ is 50 to 150 mg/kg; The reference dose of tetraoxypyrimidine (alloxan) is 60 to 110 mg/kg.

STZ is a nitroso-containing compound that enters the body specifically destroys islet β cells by:

(1) STZ directly destroys islet β cells: mainly after injection of high doses of STZ. STZ injection can cause β decrease in the concentration of intracellular coenzyme I (NAD), NAD-dependent energy and protein metabolism to stop, resulting in β cell death;

(2) Destruction of islet β cells by inducing the synthesis of nitric oxide (NO);

(3) STZ activates the autoimmune process, which further leads to damage to β cells: small-dose injection of STZ can destroy a small number of islet β cells, dead islet β cells can be engulfed by macrophages as antigens, producing TH1-stimulating factors, making TH1 cell lines dominant and producing IL-2 and IFN-γ, promoting inflammatory cell infiltration in the islets, and activating and releasing IL-1, TNF-α, IFN-γ, NO and H2O2 and other substances to kill cells. Dead cells, in turn, can act as autoantigens and are re-presented to antigen-presenting cells for processing, releasing cytokines, amplifying cell damage effects, and eventually inducing DM.

After entering the body, tetraoxaline can be rapidly uptake by islet β cells, affecting the permeability of cell membranes and the production of intracellular ATP, inhibiting glucose-mediated insulin secretion. Tetraoxypyrimidine mainly destroys the cell structure of β by producing oxygen radicals, resulting in cell damage and necrosis, thereby hindering the secretion of insulin and reducing serum insulin levels. Because tetraoxopyrimidine causes diabetes mellitus and causes toxic damage to liver and kidney tissues, and some animal models of DM made of tetraoxypyrimidine can spontaneously alleviate, it has been rarely used.

3. Animal models of spontaneous diabetes

The model was mostly molded using inbred purebred animals with spontaneous DM tendencies, such as BB (Biobreeding) mice, NOD (non-obesity diabetes), G (Goto-kakisaki) rats, and Chinese gophers (Chinese hamster). Spontaneous DM animal models refer to animal models in which the animal undergoes DM under natural conditions without any conscious artificial disposal. There are about 20 species of spontaneous DM animals that have been used for research, which can be divided into two categories: one is insulin deficiency, rapid onset, symptoms are obvious, and accompanied by ketoacidosis, such as BB (Biobreeding) mice, NOD (non-obesity diabetes) mice, and LETL rats, which can be used as animal models of type 1 DM. These animals were not obese, presented symptoms of pancreatitis at the beginning of the disease, and were involved in the pathogenesis of human tissue-associated antigens (MHC), which were similar to the characteristics of human type 1 DM. Using these models, the pathogenesis of human type 1 DM can be studied in depth; The other type is insulin-resistant hyperglycemia, which is characterized by a long course of disease, no ketoacidosis, and is an animal model of type 2 DM. Commonly used animal models of type 2 DM spontaneous animals are Chinese gopher (Chinese hamster), GK (Goto-Kakisaki Wistar rats), NSY (Nagoya-Shibata-Yasuda) rats, and OLETF rats.

(1) Animal model of type 1 diabetes

1. BB rats

BB rats are commonly used type 1 DM model animals and were bred by the Biobreeding Laboratory in Ottawa, Canada. DM occurs in about 50% to 80% of BB rats, with males and females having a similar incidence. BB rats generally develop DM at age of 60 to 120 days, and glucose intolerance and isletitis may be seen a few days before the onset of disease. Onset rats have the typical characteristics of type 1 DM: weight loss, polydipsia, polyuria, diabetes, ketoacidosis, hyperglycemia, hypoinsulination, isletitis, islet β cytopenia, and require insulin therapy to survive. Another feature of BB rats is that they have lymphocytes in their blood and are susceptible to infection. In addition, lymphocytic thyroiditis occurs more frequently in BB rats, and their serum often detects autoantibodies against smooth muscle, skeletal muscle, anti-parietal cells, and antithyroglobulin.

2. NOD mice

NOD mice are a kind of spontaneous non-obese DM mice, its age of onset and incidence have obvious gender differences, female mice have a significantly earlier age of onset than male mice, the incidence is much higher than that of male mice, NOD mice begin to appear isletitis at 3-5 weeks of age, infiltrating islet lymphocytes are often CD4+ or CD8+ lymphocytes, and significant DM occurs at 13 to 30 weeks of age. Unlike BB rats, NOD mice generally do not develop ketoacidosis and do not have peripheral hemolypolysis, but also require insulin therapy to maintain survival. In the early stage of NOD rat isletitis, the basic value of insulin and the response value to glucose in plasma and islet perfusion fluid were reduced, while the immune activity of glucagon and glucagon-like substances increased, the activity of glucose kinase and pyruvate kinase in NOD mice decreased, the activity of glucose-6-phosphate dehydrogenase and pyruvate kinase increased, transaminases, lactate dehydrogenase, branched-chain amino acids and β-N-acetamidurazine in kidney tissues, Activities such as α-glucosidase and α-mannitosidase were also reduced. NOD mice with DM are the result of a multifactorial combination of genetic, immune and free radical damage, and these characteristics of NOD mice are similar to those of patients with type 1 DM, and are good animal models for studying the genetics, immunology, virological characteristics of type 1 and their prevention and treatment.

3. LETL (long evans tukushima lean) rat

LETL is also an animal model of DM type 1, usually developing DM at 8 to 20 weeks of age, with an incidence of about 21% in male rats and about 15% in female rats, and if rats treated with cyclophosphamide at 5 to 7 weeks of age, their DM incidence doubles at 16 weeks of age. LETL rats have no peripheral hemolypolysis, and significant lymphatic infiltration can be seen in the islets 4 to 5 days before the onset of obvious DM symptoms.

(2) Animal model of type 2 diabetes

1. Psammonys Obesus (PO)

PO rats are rodents living in desert areas, the rat has significant insulin resistance, under a high-calorie diet (a few days to two weeks), 90% of PO rats spontaneously develop hyperinsulinemia, accompanied by significant hyperglycemia, followed by a decrease in insulin levels. The onset of DM in PO rats can be roughly divided into the following four stages: (1) The initial stage: the blood glucose and serum insulin level in this stage are normal; (2) Hyperinsulinemia: the blood glucose remains normal in this period, but the serum insulin is significantly elevated; (3) Hyperinsulin and hyperglycemia period: the blood glucose > during this period is 11.1mmol/L; (4) Hypoinsulinic hyperglycemia period: In this period, due to the damage to the secretion function of islet β cells, low insulin and hyperglycemia are caused, rats need to be treated with insulin to maintain survival. Duhault et al. found that PO rats were insulin-dependent in the late stage of type 2 DM, and pancreatic histology showed the presence of isletitis, indicating that it had the characteristics of late-onset type 1 DM (Latent autoimmune diabetes mellitus in adult, LADA), so PO rats may be suitable for LADA research.

2. Chinese gopher

The spontaneous DM gopher model is obtained by inbreeding healthy Chinese gophers. This model is characterized by mild and moderate hyperglycemia, the animals are non-obese, the serum insulin performance is diverse, and the degree of islet lesions varies, similar to human type 2 DM. Most gopher DM incidence is within 1 year of age, and the population incidence is about 20.88%.

3. GK rats (Goto-Kakisaki Wistar Rats)

GK rat is a commonly used spontaneous non-obese type 2 DM model animal, and the incidence of GK female and male rats is comparable, and the OBVIOUS DM generally occurs at 3 to 4 weeks of age. Before hyperglycemia occurs, there is often a period of normal blood glucose (from birth to weaning), which is equivalent to the pre-DM period in humans. It is characterized by: impaired secretion of glucose-stimulated insulin, a 60% decrease in the number of β cells, a decrease in the sensitivity of the liver to insulin, resulting in excessive hepatic glucose production; Muscle and adipose tissue are moderately insulin resistant. GK rats also had higher blood pressure than normal Wistar rats (about 15 mmHg higher for low salt and 24 mmHg higher for high salt). In addition, GK rats had changes similar to those of human type 2 DM microvascular complications such as slowed motor nerve conduction rate, segmental demyelinating of nerve fibers, axial mutantness, increased expression of retinal vascular endothelial growth factor (VEGF), decreased local blood flow to the retina, albuminuria, thickening of the glomerular basement membrane, glomerular hypertrophy and hardening.

4. Zucker DM obese (zucker diabetic fatty, ZDF) rats

ZDF rats are commonly used type 2 DM model animals, which lead to hyperphagia and obesity due to leptin receptor mutations, accompanied by hyperinsulinemia, hyperlipidemia, and moderate hypertension. Male ZDF rats generally develop DM at 8 to 10 weeks, with typical symptoms of DM such as polydipsia, polyuria, and slow weight gain, and may develop neuropathy. Muscle GLUT4 expression in male ZDF rats was significantly reduced, and glut2 expression in islet β cells was also significantly downregulated, which may be the mechanism of type 2 diabetes in ZDF rats. Histological studies have found that the islets of 6-week-old rats exhibit structural disorders and fibrosis, islet β cell degranulation, and the number of islet β cells is much lower than that of non-DM ZDF rats of the same week age.

5. NSY (Nagoya-Shibata-Yasuda) mice

NSY mice are produced from jcl ICR orthogonal mice based on glucose tolerance selection and are characterized by age-dependent spontaneously occurring DM. The rats did not have severe obesity at any age, nor did they have high levels of hyperinsulinemia, but at 24 weeks of age there was a significant weakening of glucose-stimulated insulin secretion. Morphological abnormalities such as islet hyperplasia or inflammatory changes were not seen in pathology, suggesting that the cause of type 2 DM in NSY mice may be the change in glucose-induced insulin secretion function of islet β cells. Insulin resistance may play a role in its pathogenesis. NSY mice will contribute to further research on the genetic predisposition and pathological occurrence of type 2 DM.

6、 OLETF(Ostuka Long-Evans Tokushima Fatty)大鼠

The OLETF rat is a spontaneous type 2 DM model animal established by Kono et al. using Long-Evans rats. Due to the complete deletion of the expression of gallbladder indentin (CCK)-A receptor mRNA, the mouse was hyperinvested and obese, the digestive tract did not respond to CCK-8 stimulation, and the endocrine function of the pancreas was reduced. This model has the characteristics of type 2 DM such as polyphagia, obesity, polydipsia, and polyuria, and can slowly and naturally produce type 2 DM. From the age of 8 weeks, the serum triglycerides, cholesterol and postprandial blood glucose were significantly higher than those of the control mice, and the serum triglycerides and postprandial blood glucose continued to increase with age. Significant insulin resistance appears from 12 weeks of age; At 18 weeks of age, insulin sensitivity is about 20% of that of the control group; At 24 weeks of age, plasma insulin compensatory increases; At 30 weeks of age, the blood TG level reached 5 times that of the control group; After 40 weeks of age, the secretory function of the islets decreases; After 65 weeks, blood glucose levels were as high as 25 mmol/L, while immunoreactive insulin (IRI) levels were below 40 pmol/L. OLETF rat urine protein increased significantly from 30 weeks of age, and increased rapidly with age, male rats 55 weeks old, urine protein content can reach more than 800mg / day. Histological studies have found progressive fibrosis of the pancreas in OLETF rats. At 20 weeks of age, the pancreas has obvious fibrosis and islet enlargement; At 40 weeks of age, the islets are replaced by connective tissue; At 70 weeks of age, the pancreas is extremely atrophied and pancreatic tissue is replaced by fat and connective tissue. In addition, OLETF rats can develop glomerular basement membrane thickening at 22 weeks of age; After 40 weeks of age, male OLETF rats have enlarged glomeruli, glomerular membrane stromal hyperplasia and glomerular basement membrane thickening; At 70 weeks of age, PAS-positive nodules surrounded by dilated capillaries are visible around almost every glomerular, and this nodular change swells into the glomerular membrane matrix, and the kidney changes in OLETF rats are very similar to human DM nodular glomerulosclerosis. The above pathological changes in different stages of pancreas and kidneys are very similar to the pathological manifestations of clinical type 2 DM patients, which provides a good experimental animal model for studying the pathogenesis of type 2 DM and its complications and the evaluation of insulin resistance interventions.

7. Db/db mice

Diabetes mellitus in db/db mice is caused by mutations in leptin receptors and is autosomal recessive. The rat developed polyphagia and hyperinsulinemia at 10 to 14 days of age, but the blood glucose remained normal at 4 weeks of age, and then the rat gradually increased in weight and hyperglycemia. Although insulin levels are 6 to 10 times normal at 2 to 3 months of age, blood glucose levels can reach 22 to 33 mmol/L. Insulin levels gradually decreased below normal levels at about 3 to 6 months of age, during which the mice lost significant weight and developed ketonia, histology showed significant β cell necrosis, and the mice survived for no more than 10 months if insulin therapy was lacking. Another feature of db/db mice is that their serum glucagon levels are more than 2 times higher than those of normal controls. Db/db mice are an animal model suitable for studying the pathogenesis of type 2 diabetes.

8. Ob/ob mice

Ob/ob mice are type 2 diabetes model animals that are autosomal recessive. The onset of diabetes in ob/ob mice is due to mutations in the ob gene, resulting in deficiency of the protein leptins encoded by them, causing a significant increase in hepatic steatogenesis and hepatic glycogens, and hyperglycemia stimulates insulin secretion, causing insulin resistance and stimulating the formation of fat. Ob/ob mice can weigh up to 90 grams. The severity of symptoms in ob/ob mice depends on the genetic background. Homozygous animals present with obesity, pronounced hyperglycemia, and hyperinsulinemia. Insulin levels in ob/ob/6J mice can reach 10 to 50 times that of normal mice, but their blood glucose is often only mildly elevated. Histology showed that the islet β cells in ob/ob mice were significantly hyperplastic and hypertrophied, while the number of islet A cells, D cells and PP cells was significantly reduced.

9. KK rat

KK mice, a mildly obese type 2 diabetic animal, crossed with C57BL/6J mice and bred to obtain Tornto-KK (T-KK) mice. The yellow obesity gene (i.e., Ay) was transferred to KK mice, KKAy mice, which had significant obesity and diabetic symptoms compared to KK mice. KK mice have significant polyphagia, blood glucose from 5 weeks of age, blood insulin levels gradually increased, to 5 months of age weight up to 50 grams, non-fasting blood glucose is often less than 17mmol / L, non-fasting blood insulin up to 1200ug / mL; Polyphagia, hyperglycemia, hyperinsulinemia, obesity, and liver sensitivity to insulin at 1 year of age can spontaneously return to normal, but the life of diabetic KK mice is often significantly shortened. In addition, KK mice had elevated fasting glucagon levels and were not inhibited by glucose. Histology shows degranulation and glycogen infiltration of β cells, followed by islet hypertrophy and hepatic lipidization and adipose tissue increase.

4. Other DM animal models

(1) Animal model of hormonal DM: injection of anterior pituitary extract, auxin, adrenocorticoids, thyroxine or glucagon can directly or indirectly produce DM.

(2) Viral DM animal model: the use of cerebro-myocarditis virus (EMC-M virus) and coxsackie virus to degranize and necrosis of mouse islet β cells of certain species, resulting in the destruction of islet β cells and producing similar type 1 DM.

(3) Animal models of immune DM: intravenous injection of anti-insulin antibodies or Freund's adjuvant complex with isotropic or xenoin insulin and anti-serum immunization; Or immunization of animals with the same or xenopancreas + Freud's adjuvant can produce transient hyperglycemia after several hours. The mechanism is that endogenous insulin binds to the input antibody resulting in a decrease in endogenous insulin and dm.

(4) Animal model of hypothalamic DM: electrocoagulation or injection of glucose gold can damage the ventral medial nucleus (VMH) satiety center of the lower abdomen of the thalamus, which can cause mature animals to over-feed, obesity, and even DM.

5. Animal model of transgenic diabetes

Transgenic animal technology is the artificial modification or modification of the structure or composition of the animal genome by means of genetic engineering, and the corresponding animal breeding technology enables these modified genomes to be transmitted and expressed between generations. Using this technology, people can introduce designed genetic mutations at specific sites in the animal genome, simulating the genetic structure or number of abnormalities that cause human genetic diseases. By modifying the gene structure, the function of genes in vivo and the relationship between their structural functions can be studied in the whole process of animal occurrence and development. Genetically modified DM animals with type 1, type 2 DM and adolescent-onset diabetes of youth (MODY) have been reported.

(i) MKR mouse animal model

MKR mice are transgenic animal models of type 2 DM bred by Fernández et al. The mouse skeletal muscle overexpressed the inactivated IGF-1 receptor, and the inactivated IGF-1 receptor formed a hybrid receptor with the endogenous IGF-1 receptor and insulin receptor, interfering with the normal function of these receptors and leading to significant insulin resistance. The mice had obvious hyperinsulinemia at 2 weeks of age, gradually increased blood glucose on fasting and after eating after 5 weeks of age, and had obvious glucose tolerance abnormalities at 7 to 12 weeks of age. Treatment with PPAR-agonists WY14, 643 corrects abnormal glucose metabolism in MKR mice.

(ii) MODY2 animal model

Zhang et al. used gene knockout technology to obtain liver glucoskinase (GCK)-mice, and found that with the prolongation of time, the mouse gradually increased blood glucose, glucose tolerance decreased, and the fasting blood glucose of 6-week-old mice was significantly higher than that of control mice, and the disease caused by the decreased activity of hepatocyte GCK was similar to that of human MODY, which can be used as an animal model of MODY.

(c) Mitochondrial diabetes

Silva et al. found that mice with mutations in β cells Tfam (mitochondrial transcriptionfactor A) developed diabetes mellitus at about 5 weeks of age, manifested by severe mtDNA depletion, insufficient oxidative phosphorylation, and abnormal mitochondria observable in the islets at 7 to 9 weeks, showing changes in mitochondrial diabetes.

(4) GDM (Gestational Diabetes) Model

Insulin resistance during pregnancy can cause gestational diabetes mellitus, and patients with fetal characteristics are macrosomia, with an increased probability of obesity and type 2 diabetes in adulthood. Yama***a et al. found that the heterozygous C57BL/PKsJ-db/+ mice developed GDM. Pregnancy causes the dissociation of PI3K from IRS-1, increased activity of binding to IR, increased insulin-mediated arginine phosphorylation and decreased expression of IRS-1 and its tyrosine phosphorylation, resulting in a decrease in the ability of IRS-1 to bind and activate PI3K, the inability of insulin function to function, and insulin resistance to occur [).

(5) In order to study the role of B lymphocytes in the pathogenesis of type 1 DM, Wong et al. used transgenic technology to prepare NOD-RIP-B7-1 (Nonobese Diabetic-Rat Insulin Promoter-B7-1) transgenic mice, which caused the onset of DM to be significantly earlier than normal NOD mice due to overexpression of auxiliary stimulator B7.1, and DM occurred at 12 weeks.

With the gradual deepening of DM research, the corresponding development of DM animal models is inevitable, and the establishment of transgenic animals will provide more scientific and effective tools for DM research.