Douglas L. Coleman

Laboratory animals exhibiting obesity and diabetes syndromes.

Spontaneous hyperglycemia, hyperinsulinemia and obesity are common features for at least one period of the lifetime in some strains of mice. Both genetic and environmental factors are involved in the pathogenesis of the diabetes-like syndrome, making these strains excellent models for studies in both obesity and diabetes-like states. The metabolic peculiarities can be due to a dominant gene, as for the yellow obese, or a single recessive gene, as in the obese and the diabetes mouse; or they can be of polygenic origin, as for the KK and the NZO mouse. However, the severity of the metabolic disorder is due to the interaction of the mutant genes iwth modifiers in the bat genes themselves. Studies on the pathophysiology and biochemistry of these animals have revealed interstrain differences, different patterns of development of the metabolic disorder, and different degrees of severity of the diabetes-like syndrome. Although the primary causes of the syndrome remain unclear in some strains, an involvement of hypothalamic feeding centers has been implicated.

Effect of diet on incidence of diabetes in nonobese diabetic mice.

Nonobese diabetic/Lt mice exhibit a diabetes incidence >70% in females at 30 wk of age. In studies designed to see whether increased dietary carbohydrate, fat, or protein influenced the severity or age at onset of the syndrome, we fed semipurified AIN-76 diet adulterated with increased amounts of these ingredients. Surprisingly, all AIN-76–based diets greatly reduced the expected incidence of diabetes at 30 wk. In addition, a hypoallergenic infant formula, Pregestimil, containing casein hydrolysate in place of protein, completely prevented diabetes up to 1 yr of age. To assess how dietary components might modulate the diabetes incidence, we adulterated standard AIN-76 diet with skim milk, gluten, brewers yeast, or a natural-ingredient rodent open-formula mouse diet (Old Guilford 96 [OG96]). No increase in diabetes incidence was seen with skim milk (10%) or wheat gluten (10%), whereas brewers yeast (10%) and OG96 (25%) added to AIN-76 increased the incidence compared to mice fed OG96 only. The diabetogenic factor or factors in OG96 could be extracted by chloroform plus methanol (2:1), leaving little activity in the residue. We conclude that diet is a critical factor in diabetes development and that unknown chloroform-methanol–soluble substances in natural-ingredient chow not found in semipurified diets can enhance the development of diabetes in genetically susceptible mice.

The Influence of Genetic Background on the Expression of Mutations at the Diabetes Locus in the Mouse: III. Effect of H-2 Haplotype and Sex

The expression of the mouse mutation, diabetes (db), was examined on eight different inbred genetic backgrounds. The influence of H-2 haplotype and sex was examined. Mice of both sexes in two diabetes (db) strains (C57BL/6J, 129/J) having the H-2b haplotype were resistant to the diabetogenic action of the mutant gene. On the contrary, two H-2d congenic diabetes stocks (C57BL/KsJ, DBA/2J) exhibited severe diabetes associated with beta-cell necrosis. However, diabetes resistance was not restricted to mice with H-2b haplotype since the congenic diabetes MA/J stock (H-2k) was also resistant. Similarly, diabetes susceptibility was not restricted to mice with the H-2d haplotype, since males, but generally not females, in the congenic CBA/Lt-db/db and C3HeB/FeJ-db/db stocks (both H-2k) also exhibited a severe diabetes. Males of the congenic SWR/J-db stock (H-2q) had a diabetes of intermediate severity. Female diabetes mice with H-2k and H-2q haplotypes exhibited a sustained hypertrophy and hyperplasia of beta-cells and were able to control hyperglycemia better than males. Thus, while the H-2b haplotype remains associated with resistance, and the H-2d haplotype with susceptibility to induction of genetic diabetes, the diabetes stocks with H-2k haplotype clearly illustrate the importance of non-H-2, but sex-associated, genetic modifiers.

Age- and Diabetes-related Changes in Tissue Glucose Uptake and Estradiol Accumulation in the C57BL/KsJ Mouse

The effect of the diabetes (db/db) mutation on the agerelated changes in glucose uptake and estradiol incorporation in peripheral tissues were investigated in C57BL/KsJ mice between 2 and 16 wk of age. Glucose uptake in the uterus, ovaries, pancreas, lung, liver, heart, kidney, and spleen were markedly increased in diabetic mice after the development of the hyperglycemic condition, as compared with control mice. The age-related increase in glucose uptake observed in control mice was enhanced in hyperglycemic (i.e., ≥4 wk of age) animals. In contrast, the diabetes mutation caused a decreased estradiol uptake by the uteri, ovaries, and mesometrial fat pads at 16 wk, while having little effect in nontarget tissues of diabetic mutants. These data indicate that the diabetes mutation enhances glucose uptake, especially in estradiol target tissues (i.e., uterus, ovary), at the same time that estradiol incorporation is depressed. These results suggest that an alteration in glucose utilization by steroid-sensitive reproductive tract tissue may underlie the impaired reproductive ability in these animals. Other peripheral tissues did not demonstrate any remarkable changes in estradiol uptake, but the enhanced carbohydrate metabolism observed may relate to the subsequent age- and diabetes-related changes in tissue structure and function in these animals.

Metabolic and Underlying Causes of Diabetes Mellitus

It is emphasized that animai models should be used to study specific genotypic or phenotypic expressions associated with diabetes rather than assuming a single animal model can reflect diverse forms of the human disease. Diabetic and normal animals are reviewed on the basis of their usefulness as models of genetic, viral, and chemically induced diabetes, including the often associated immune phenomena. Characteristics of spontaneously diabetic animals with and without obesity are also described with an emphasis on both genetics and metabolic derangements. Recommendations for future animal experimentation include: more longitudinal studies evaluating the role of sex, prenatal environment, diet, and viral or chemical attack on B-cell function; characterization of the immune phenomena associated with B-cell lesions (and insulitis) in diabetic and immunologically incompetent lines; clarification of relationships between obesity and islet-ceil function with emphasis on the role of fuel metabolism, vitamins, and minerals; and, finally, the development of new models with specific genetic aberrations placed in normal or diabetic lines.

Susceptibility to db gene and streptozotocin-induced diabetes in C57BL mice: control by gender-associated, MHC-unlinked traits

H-2 haplotype differences distinguish the related C57BL/KsJ (BKs) and C57BL/6J (B6) inbred strains. BKs mice are more susceptible to diabetes induction by a recessive obesity gene, diabetes (db), or by multi-dose streptozotocin (MSZ) administration. The purpose of this study was to evaluate whether the H-2 differences were the important genetic background modifiers determining inbred strain susceptibility or resistance to these diabetogenic stresses. Diabetes susceptibility of BKs.B6-H-2b congenic mice was compared with that of the parental BKs and B6 stocks. In addition, diabetes severity was studied in (B6 × BKs)F1 and F2db/db mice and an H-2 segregation analysis was performed. BKs susceptibility genes expressed in a dominant fashion in the F1 generation, and were transmitted to F2db/db males without apparent segregation. No association between H-2b haplotype and B6-type diabetes resistance was found in response to either the db mutation or to MSZ. Insulitis, associated with development of hyperglycemia in BKs males, also occurred in the H-2b congenic stock. However, an apparent interaction between H-2b haplotype, the db mutation (on chromosome 4), and male gender (Y chromosome?) was indicated by a segregation ratio distortion in recovery of this genotype. A more moderate diabetes in some F2db/db females suggested that non-MHC-linked genes controlling sex steroid metabolism were the important determinants of diabetogenic sensitivities in the C57BL stocks. In support of the latter, strain differences were demonstrated in activity levels of steroid sulfatase, which is regulated by a sex-linked gene likely expressed on both the X and Y chromosome, and which may control tissue levels of active androgens and estrogens. We show that the diabetes-susceptible F1 hybrids exhibit the higher activity associated with the BKs strain.

A new mutation (db3J) at the diabetes locus in strain 129/J mice. I. Physiological and histological characterization.

SummaryA spontaneous recessive mutation appearing in strain 129/J mice at the diabetes (db) locus on Chromosome 4 has been characterized. The new allele, designated db3J,produced hyperphagia and severe obesity. Mutants weighed in excess of 70 g by 6 months of age, compared to 22–28 g for lean littermates. Although the disease was similar to the mild hyperglycaemia-severe obesity syndrome exhibited by db gene presentation on the C57BL/6J inbred background, the syndrome in 129/J mice reduced lifespan, with mutants exhibiting sudden weight loss, hypoglycaemia, and a 67% mortality between 6 and 14 months of age. Mutant males, but not females, were transiently hyperglycaemic between 2 to 4 months of age, attaining a maximum mean blood sugar of 196±27 (SEM) mg/dl. Thereafter glucose levels declined to normoglycaemic values (80–100 mg/dl), and with increasing age, mutants of both sexes became hypoglycaemic (60 mg/dl at 9 months). Mutants of both sexes were extremely hyperinsulinaemic at the earlier ages, with mean plasma insulin at month 5 reflecting 30-fold elevations above normal for males and 18-fold for females. These levels diminished with age, the decline being more marked in males. Plasma glucagon levels were 3-fold elevated in the younger mutants of both sexes (86 pg/ ml versus 28 pg/ml in normal mice), mean levels increasing to almost 5-fold above mean control values in the older age group (198 pg/ml versus 41 pg/ ml in normal mice). Histopathological findings were limited to pancreas. Increasing necrosis of the exocrine, but not endocrine, pancreas was noted in aging mutants. Aldehyde fuchsin staining of the mutant pancreas revealed hyperplastic islets filled with heavily granulated B-cells. B-cell hyperplasia was accompanied by a 30-fold increase over controls in pancreatic insulin content in the 8 month old mutants, whereas pancreatic glucagon content was only doubled. Morphometric analysis showed less than a 2-fold increase in the mean number of A-cells per islet. Thus, an interesting feature of expression of the diabetes gene in the 129/J strain is the persisting hyperglucagonaemia in the face of moderating hyperinsulinaemia.

Morphometric analysis of medial basal hypothalamic neuronal degeneration in diabetes (db/db) mutant C57BL/KsJ mice: Relation to age and hyperglycemia

Age- and diabetes-related neuronal degenerative changes were morphometrically evaluated in the arcuate (ARC) and ventromedial (VMH) hypothalamic nuclei of control (+/?) and diabetic (db/db) C57BL/KsJ mice between 4 and 16 weeks of age. By 4 weeks of age, (db/db) mice exhibited marked obesity and hyperglycemia relative to controls. An increase in the population of degenerated ARC neurons was detected at 8 weeks of age in (db/db) mice relative to (+/?) animals. By 16 weeks of age, a significant increase in the number of degenerated VMH neurons in (db/db) mice was found, relative to controls. In addition, the neuronal density (neurons/mm2 area of nucleus) of both the ARC and VMH nuclei was found to be depressed in (db/db) mice, relative to controls, by 16 weeks of age. These data suggest that the normal degenerative loss of ARC and VMH neurons that occurs with age in normal mice is enhanced in the (db/db) mouse. These findings suggest that a functional alteration in hypothalamic nuclei which are recognized to modulate autonomic, pancreatic and pituitary activity may be associated with the onset or expression of the diabetic condition in the C57BL/KsJ (db/db) mouse.

Lessons from studies with genetic forms of diabetes in the mouse.

Genetically defined animal models of diabetes have many advantages over models in which the genetic component has not been established. Such models permit predictable numbers of normal and afflicted animals, differing by only a single gene, to be produced at will. Maintenance of these individual mutations in inbred strains of mice permits an evaluation of any gene-host interactions that act by modifying the severity of the diabetic condition. These genetic models provide precision tools for research in which the mutant gene itself, the inbred background, and the environment can be manipulated at will. In addition there is sufficient knowledge about the arrangement of individual genes in chromosomes in the mouse to permit one to identify, and use, closely linked markers in order to predict with confidence the mice destined to become diabetic. Such studies on the preclinical stages are of utmost importance and cannot be undertaken conveniently in any other model. Our studies with genetic mouse models have established that there are at least six genes in the mouse that can cause diabetes and obesity syndromes. The severity of the diabetes produced depends on the interaction of the mutant gene with the host inbred background as well as with other environmental factors. Establishing the nature of these interactions and the possible primary lesions involved in each genetic syndrome should have major ramifications in studies dealing with human diabetes.

Insulin secretion in experimental obesity

Abstract In mice rendered obese by adminitration of goldthioglucose, the pancreas increases both its store of insulin and its capacity to secrete insulin in vitro in response to glucose and theophylline in direct proportion to the actual body weight. In hereditarily obese mice ( ob ob ), the insulin stores and secretory capacity of the pancreas increase even more, so that, when related to body weight insulin content and output average respectively 3 and 6 times the mean values found in lean mice. An increase in the amount of peripheral tissues to be supplied with insulin and some extrapancreatic factor are considered as possibly involved in the changes of islet function seen in experimental obesity.