An animal model for senescence has been developed during these 20 years at Dept. of Senescence Biology, Chest Disease Research Institute, Kyoto University. The characteristics of ageing dynamics in this model is designated as accelerated senescence, so this model is named “Senescence Accelerated Mouse (SAM) .” SAM consists of 2 series; SAM-P (accelerated senescenceprone mouse) and SAM-R (accelerated senescence-resistant mouse) . The former are SAM-P/1, -P/2, -P/3, -P/4, -P/6, -P/7, -P/8 and the latter are SAM-R/1, -R/2 and -R/3.
Senescence phenomena are accelerated in the SAM-P series and characterized by a shorter life span and early manifestation of various signs of senescence, including alteration of physical activity, loss of hair glossiness, increasing coarseness of the hair and the hair loss, periophthalmic lesions, increased lordokyphosis of the spine and so on, after a normal process of development. Characteristic pathologies closely associated with senescence observed in SAM are senile amyloidosis in SAM-P/1 and -P/2, senile cataract in SAM-P/3 and SAM-R/3, senile osteoporosis in SAM-P/6, deficits in learning and memory in SAM-P/8 and degenerative arthritis in SAM-P/3. The murine model SAM will be a valuable tool to clarify the pathogenesis of age-associated diseases and also to reveal the basic mechanism of ageing. The genetic background related to development of this model is discussed.

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(1) A model for the biological function based on the gene function.
Today, it is widely known that various strains of B10.H-2 congenic mice established by Dr. Snell in the Jackson Laboratory have played an important role in the remarkable breakthrough of the modern immunogenetics, not only in mouse but in man. Of course, man diverged genetically from mouse by several ten million years, while the basic biological principle, immunological one in this case, is obviously common between them. Thus, H-2 congenic mice have made a great contribution to the biomedical sciences as not a disease model but a model for the biological function.
Homology between man and mouse in their gene arrangement on the sizable length of chromosome has recently been recognized as ‘Synteny.’ This has also given more valuable status to the mouse than before, being accompanied with the technical breakthrough in gene manipulation. It became possible to find out a mouse gene corresponding to a human gene of interest from a viewpoint of ‘Biological function model.’
(2) Genetic differentiation of house mouse species and the status of laboratory mice.
Though quite a few genetic variations have been found in the laboratory mice so far, those are still not enough for the further development of the modern biomedical research. Since 1975, we have investigated genetic differentiation of Mus musculus subspecies in the world. A considerable genetic distance up to one million years was able to be estimated between each major subspecies, European M. m. domesticus, Asian M. m. musculus, M. m. bactrianus and M. m. castaneus. The allelic constitutions in the wide variety of the present laboratory mouse strains are almost similar to those of domesticus subspecies, strongly suggesting that a possible origin of those laboratory mice is domesticus-derived fancy mice in Europe.
(3) A variant gene introduced from Japanese wild mouse, being useful for the study of basic biological functions.
More than ten B10.MOL⋅H-2 congenic strains which carry the Japanese wild-derived H-2 chromosome in B10 background, have been established by us. One of them, B10.MOL-SGR strain, exhibited the tremendously higher meiotic recombination within the H-2 complex, in particular between K and A genes. This recombinational hot spot was assigned within 1 Kb of this region. The DNA fragment containing it was cloned and sequenced. Apparently, this novel wild-derived congenic strain can greatly contribute to further understanding of the molecular mechanism of meiotic recombination which is obviously quite basic biological function.
(4) A model mouse for 21 hydroxylase deficient trait in man.
Probably due to the extraordinarily higher recombination frequency in the H-2 complex mentioned above, B10.MOL-SGR strain emerged a new mutant carrying “aw18” haplotype. The homozygous mice for this H-2 complex died at 5-10 days of age by the steroid hormone disorder which was caused by the deletion of 21-hydroxylase gene in the H-2 complex. This could be an excellent model for the 21 hydroxylase deficient patient in man. We are now trying to rescue this lethal trait by the introduction of the normal allele as a transgene.

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The NOD mice spontaneously develop insulin-dependent diabetes mellitus characterized by autoimmune insulitis. They have unusual class II MHC: the expression of I-E molecules are clearly absent and I-A molecules have unique characteristics. To determine whether the unusual expression of the class II MHC may be responsible for the development of insulitis in this strain, two series of experiments have been attempted utilizing transgenic mice carrying MHC class II genes.
1) By mating NOD mice with I-E expressing C57BL/6 (Eα^d) transgenic mice, backcross progenies which expressed I-E molecules without having other MHC genes on chromosome 17 derived from C57BL/6 were obtained. No insulitis was observed at 25 weeks of age in these backcross progenies.
2) NOD transgenic mice carrying Eα^d or Aβ^k gene were directly produced by microinjecting these genes into fertilized NOD eggs. I-E or I-A^d/k antigens were detected on the surface of their lymphocytes. In NOD (Eα^d) transgenic mice, no insulitis was observed at 19 weeks of age. On the other hand, in 4 among 5 NOD (Aβ^k) transgenic founders expressing I-A^d/k antigens developed insulitis until 19 weeks of age. These experiments clearly demonstrate that one of the diabetogenic recessive gene linked to the MHC locus on chromosome 17 is Eα gene itself deleted in NOD mouse.

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The KK line of mice, established by Kondo et al. in 1957, is spontaneously obese, hyperinsulinemic, and often hyperglycemic; these mice are used as a model for non-insulin dependent diabetes mellitus. Although KK mice vary considerably in body weight, and blood and urinary glucose levels, they are resistant to the hypoglycemic action of insulin and intolerant to glucose. However, KK mice are predisposed to diabetes in response to the onset of obesity, induced by injection of goldthioglucose, feeding of high energy diets, or introduction of the yellow obese gene (A^y) . This predisposition is considered to be genetically determined resistant to insulin, as evidenced by impaired sensitivity of adipocytes and muscle to insulin. Obese KK mice are characterized by renal glomerular lesions consisting of a diffuse or exudative type of sclerosis, accompanied by albuminuria. These changes, though less remarkable, are observed also in non-obese KK mice. These findings indicate that KK mice are useful to evaluate new anti-diabetic and anti-obesity agents designed to improve insulin resistance and obesity-related metabolic disturbances. In the present symposium, I would like to present characteristics of KK mice, and their responses to ADD-3878, an agent to improve insulin resistance and AO-128, an intestinal disaccharidase inhibitor.

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The NON mouse, a subclone of the NOD mouse, has been found to be non-obese, glucoseintorelant with a mild hyperglycemia. However, little is known about insulin secretion profile of the NON mouse. To elucidate the mechanisms of defective insulin secretion of the mouse, glucose tolerance test and in vitro study using isolated pancreatic islets of the mouse were carried out.
After intraperitoneal glucose loading, male NON mice exhibited a marked hyperglycemia, though their fasting blood glucose levels remained normal. The hyperglycemia after glucose loading was more marked in the “glucose” group which was given 10% glucose daily instead of tap water.
In vitro study revealed that glucose-induced insulin release from the isolated islets was blunted in NON mice. Insulin release, however, was well preserved or even exaggerated when arginine, forskolin, or BAY K 8644 were used as a secretagogue. The islet insulin content was slightly but significantly decreased in NON mice as compared with control ICR mice.
These results demonstrate that in the NON mouse an insensitivity of pancreatic B cells to glucose may be responsible for the reduced insulin secretion and hyperglycemia. A selective unresponsibleness of insulin secretion to glucose can also be seen in human NIDDM.

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Spontaneous diabetic mice (NSY: Nagoya Shibata Yasuda mice) used in the present experiment were selective breeding of JCL/ICR mice (supplied by Shionogi Pharmaceutical Co., Abrahi Laboratories) . They are of the same origin as NOD mice. The C-band chromosome analysis made by Dr. Moriwaki, however, shows that NSY mice are clearly different from NOD mice in the genetic background. This NSY mice are considered the model animals for NIDDM (non insulin dependent diabetes mellitus) from some endocrinological viewpoint.
Results: 1. Histologic findings of kidney in NSY mice: We found that about the glomerular tissue under a light microscope in NSY mice, an increase in mesangial cell and matrix is observed. This finding was corroborated by electron microscopic observations. And an increase of basement membrane-like material in the mesangial area was seen. The fluorescent pattern with FITC labelled rabbit anti-mouse IgG was observed mainly in the mesangial pattern and partially in the capillary pattern. Fluorescent patterns similar to those with IgG were obtained with IgA and IgM as well. These fluorescent patterns increased in intensity with aging.
Since immune globulin settles in the glomerulus as mentioned earlier, electron microscopic observations were made in more details. As a result, the massive dense deposits were noted in mesangial area. 2. Immunological study: 1) Lymphocyte subset of splenocyte was determined in NSY mice, NOD mice and C57BL mice by the complement dependent cytotoxicity test. As a result, acceleration of the helper/inducer system was shown in NSY mice. 2) IgG circulating immune complex (IgG-CIC) was determined in three groups mice by polyethlenglycol technique. With NSY mice, IgG-CIC significantly increased to be contrasted with control group. 3) The glomerulus of NSY mice was stained with fluorescent gp70 antibody in cooperation with Dr. Tomino (Tokai University School of Medicine) . A pattern of deposition was seen mainly along the capillary wall. This findings suggest the presence of retro-virus. This suggests a possibility of the immune complex being formed with an antibody against retro-virus.

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Spontaneously diabetic GK rats were produced from rats of Wistar strain by repeating the selective breeding using glucose tolerance as a selection indicator. Of 211 normal rats 18 were selected by oral glucose tolerance test (GTT) and by the breeding 162 first generation rats were obtained. This procedure was repeated in each generation. The mean glucose tolerance curve became more diabetic with advance of generation and all of 9th generation rats had a diabetic character. The diabetic rats are not obese and there is no sex difference as regards occurrence of severity of the diabetic state. Significantly lower insulin secretion in GTT was observed in GK rats as compared with Wistar rats. Pancreas perfusion studies revealed that GK rats lacked the first response and then showed delayed and low insulin secretion. In addition, there was significantly reduced insulin secretion in isolated GK islets as compared to Wistar rats. Histological studies showed severe deformity, atrophy and fibrosis of GK islets. These histological abnormalities progressed with age. Immunohistochemistry to insulin and glucagon displayed reduced number of B cells and malorientation of A cells within GK islets. Glycolytic pathway enzyme activities in GK livers showed significantly higher than those of Wistar rats. Insulin insensitivity in GK livers may probably suggest the existence of insulin resistance. GK rats have the perilphera neuropathy and the thickening of glomerular basement membranes. GK rat is a promising model of non-insulin dependent diabetes mellitus and a good model of diabetic complications, as well.

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In 1979, spontaneously diabetic Chinese hamsters were found in closed colony (CHA), maintained at the Department of Biology, Asahikawa Medical College. By now, diabetes mellitus has occurred at a frequency of 4.1% in CHA. Recently one diabetic subline (CHAD) that has greater than 90% incidence of glycosuria and two non-diabetic sublines (A and B) that are essen-tially free of glycosuria have been established by brother-sister mating.
Incidence of diabetes is not different in male and female in CHA and CHAD. In spite of severe hyperglycemia (over 400 mg/dl) and ketonuria, diabetic Chinese hamsters generally survive more than 12 months from the birth, without insulin injection.
In diabetic CHA and CHAD, plasma and pancreatic insulin decreased and plasma and pancreatic glucagon increased in proportion to the level of hyperglycemia.
These results agree with the histological findings of decreased B-cells and increased A-cells in islets of pancreas from diabetic Chinese hamsters.
Pathogenesis of diabetes in this animal is still unknown. In CHA, insulitis was found in 39% of animals with FPG levels 120 to 200 mg/dl. But in CHAD, insulitis was not observed before and after the onset of diabetes. On the other hand, islet cell surface antibody (ICSA) was demonstrated in animals of CHAD prior to the onset of diabetes. So classification of diabetes, Type I or II is still not clear in this animal model of diabetes.
Biochemical aspects: Total cholesterol, HDL-cholesterol, triglyceride, free fatty acid, phospholipids, and lipoperoxide levels increased in diabetic CHA, in proportion to the level of hyperglycemia. Hepatic gluconeogenic enzymes (G-6-P, Fl, 6-DP, PEPCK) increased, and glycolytic enzymes (hexokinase, pyruvatekinase) decreased in diabetic CHA and CHAD.
Insulin receptors on hepatocytes of diabetic CHA were studied. Insulin binding in diabetic animals increased mainly due to an increase in the number of receptors. And binding affinity of diabetic animals was similar to that of control animals. A significant inverse correlation between insulin binding and the plasma insulin concentration was observed in these animals.
Complications: In diabetic CHA and CHAD, kidney and glomerular volume increased from the onset of diabetes. In quantitative study, age related thickening of glomerular basement membrane was observed both in animals of strain B and CHAD. And significant increase of glomerular basement membrane thickness was observed in animals of CHAD over 8 months of age, compared with age-matched control. But nodular sclerosis or exudative lesion was rarely found even in old diabetic hamsters.
On the other hand, urinary protein excretion was significantly increased in diabetic CHAD of 4 and 8 months of age, compared with age-matched control animals. And the number of anionic sites on glomerular basement membrane, stained with cationic polyethyleneimine, was reduced in the same animals. These results suggest that a reduction of electrical property of glomerular basement membrane induces increased permeability of proteins in diabetic Chinese hamsters.
Hydronephrosis with enlarged urinary bladder was commonly observed in diabetic CHA and CHAD. Acetylcholinesterase activity was reduced in the nerves and on smooth muscle fibers in the urinary bladder of diabetic CHAD. It may suggest that pathological changes in pelvic nerves may underlie urinary bladder dysfunction in diabetic Chinese hamsters.
Glucose, sorbitol, and fructose levels were significantly increased, and inositol level was significantly reduced in sciatic nerve, lens, and retina of diabetic CHA. These changes of polyol metabolism may cause a pathophysiological changes in nerve and retina of diabetic Chinese hamsters. Another biochemical and morphological changes in bone and heart of diabetic Chinese hamsters were also reported.
In conclusion, diabetic Chinese hamster in CHA and CHAD is a model of insulin deficient diabetes

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Spontaneously developed diabetes mellitus was observed in aged males of an inbred strain of Wistar rats, WBN/Kob. Pathohistologically, at 3 months of age, inflammatory cell infiltration, hemorrhage, deposition of hemosiderin and fibrinous exudation around the pancreatic ducts or blood vessels were observed. A gradual increase in fibrous tissue was observed with advancing age. The enlarged interlobular lymphnodes were also observed. At 12 to 24 months of age, the pancreas became atrophic and replaced by the fibrous tissue and adipose tissue. At 24 months of age, deposition of hemosiderin, suggesting the past hemorrhage was also observed in extensive areas. Inflammatory cell infiltration, deposition of hemosiderin and extremely enlarged interlob ular lymphnodes in pancreas suggest the presence of persistent inflammation together with tissue injury. As the restoration of tissue injury, fibrous tissue might abundantly proliferate resulting in pancreatic fibrosis. As for endocrine, islets composed of few endocrine cells were detected and immunohistochemical study showed decreased numbers of not only B cells but also A cells. The pathophysiology of this diabetes mellitus was quite different from that of type I or II diabetes mellitus in man and animals. The WBN/Kob rat is a new strain of spontaneously developed diabetic rats with exocrine pancreatic insufficiency and also available for the spon taneously occurring chronic pancreatitis models. Moreover in this strain, insulin administration is usually not necessary for survival after the onset of diabetes and this strain is adequate for a long-term observation. As for the ocular lesions, around 1 5 months of age, opacity of lens began to appear. Opacity was first observed in the periphery of the lens, then increased rapidly in severity, extending concentrically and centripetally, until total cataracta was developed. The incidence of cataracta in male rats was gradually increased and reached almost 100% at 24 months of age. As for renal lesions, in male diabetic rats, 24 hour urinary total protein excretion began to increase at about 24 months of age and reached to 50-300 mg/24 hrs at 13 to 28 months of age, which was significantly increased compared with those in the age matched male Wistar rats. Electrophoretic analysis revealed urinary protein was almost albumin. In light microscopic examinations, glomeruli with segmental or global increase of mesangeal areas were noted at 17 months of age, developing a marked glomerulosclerosis. In electron microscopic examinations, the glomerular basement membrane in diabetic male rats aged 11 months was already thickened in comparison with that in the age matched male Wistar rats. As for neural lesions, a reduction of motor nerve conduction velocity was demonstrated in diabetic male rats. Morphometrically, diabetic rats has a remarkable decrease in density and diameter of myelinated fibers in comparison with those in control Wistar rats. From the above data, the WBN/Kob rat is also available for an animal model of human diabetic complications.

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