Variation in the severity and duration of hyperglycemia in the C57BL/6J-ob/ob (obese) mouse

The problem

The C57BL/6J obese (B6-ob/ob; 000632) mouse provides a model for analyzing the contributions of hyperinsulinemia, obesity and insulin resistance in the pathogenesis of non-insulin dependent diabetes mellitus (NIDDM) in humans. Although B6-ob/ob mice are sometimes referred to as "obese hyperglycemic," their hyperglycemia is generally observed to be transient, initiating when the mice are weaned at around 1 month of age and subsiding to a near-normal glycemic level by 5 months. This hyperglycemia is relatively mild in comparison to the chronic diabetes syndrome expressed when the same mutation is studied on the related C57BL/KsJ inbred strain background (Coleman, 1978). Investigators using B6-ob/ob mice occasionally call The Jackson Laboratory to report deviations between the expected level of hyperglycemia (based upon published literature values) versus the values they actually measure in their laboratory setting. We have recently completed a longitudinal survey of the non-fasting plasma glucose levels in a small group of current generation mutant mice and discuss these findings, along with methodological problems associated with plasma glucose determinations in mice.

Brief description of the strain

Obese (ob) is a fully penetrant and autosomal recessive obesity mutation in the proximal region of Chromosome 6. The molecular basis for the mutation remains unknown. It produces neuroendocrine perturbations that confer an unusually efficient conversion of metabolic energy into fat (Coleman, 1982; Bray, Fisler, and York, 1990). Discovered at The Jackson Laboratory in 1949, it is now propagated in a congenic strain C57BL/6J (B6-ob). Since B6-ob/ob mice exhibit derangements in a multiplicity of insulin-responsive metabolic pathways, they reflect some of the characteristics of NIDDM, and, hence, are frequently used by investigators requiring an animal model exhibiting insulin resistance and some degree of glucose intolerance. Mutant females are sexually dysfunctional. The strain is maintained by transplantation of ovaries from juvenile B6-ob/ob females into B6-+/+ hosts, which are mated to B6-+/+ males. The ob/+ heterozygotes produced from such matings are intercrossed to produce +/+, +/ob, and ob/ob mice for distribution. Homozygous ob/ob mice of both sexes are phenotypically distinguishable from their lean littermates at weaning by increased adiposity. This is associated with hyperinsulinemia already detectable in the preweaning phase. After weaning at 3-4 weeks, hyperphagia and an increasingly severe insulin resistance develop. Beginning around puberty (between 4-6 weeks of age), development of moderate hyperglycemia develops in B6-ob/ob mice, especially males. Remission from hyperglycemia is correlated with a compensatory hyperplasia and hypertrophy of the pancreatic beta cells. The resultant hyperinsulinemia is assumed to account for the remission of hyperglycemia after 12 weeks of age (Coleman, 1978).

Study description

Groups of 5 mice of each of the various genotypes described below were received from the Animal Resources Unit at approximately 4 weeks of age and segregated by sex, with each double-pen plastic cage containing a total of 5 mice. Cages and bedding (pine shavings) were changed twice weekly. Mice were allowed free access to pelleted chow (Old Guilford diet 96W, Emory Morse Co., Guilford, CT) and hyperchlorinated drinking water. The mice were kept in a specific pathogen-free, but otherwise conventional (non-barrier) research mouse room with a 14:10 light:dark cycle. Mice were weighed and plasma glucose determined at biweekly intervals. Approximately 100 l of venous blood was withdrawn in a heparinized capillary from the retro-orbital sinus of non-fasted conscious mice by an experienced phlebotomist. All collections were between 8-9 a.m. Plasma was obtained and plasma glucose determined using a precalibrated Beckman Glucose 2 analyzer. The first serum sample was not taken until the mice had acclimated to the research colony for two weeks.

Results

As expected, mutant mice of both sexes gained weight rapidly in comparison to controls (Fig.1), with males reaching a mean (± standard error) weight of 69.0 ±2.6 grams and females attaining a mean of 57.6 ±7.5 grams by 26 weeks of age. Two types of controls were compared. The designation "+/?" is used for the black lean littermate controls for ob/ob mice normally supplied by the Animal Resources Unit. This designation indicates a genotype mixture statistically expected to be composed of 2/3 ob/+ and 1/3 +/+ segregants. To assess any potential effects of the ob mutation in heterozygous state, aged matched +/+ mice from the (separate) colony of standard C57BL/6J mice maintained by the Animal Resources Unit were also studied. While the weights of 26 week old +/? and +/+ males were not different (37.1±1.1 versus 36.7±2.9 grams), the difference between the two groups of female controls was significant (29.3± 1.5 versus 23.4± 0.9 grams). This finding illustrates one of the difficulties inherent in using control mice obtained from matings different from those used to produce the mutant offspring. The +/+ females weighed a mean of 2.2 grams less than the +/? females upon receipt at 4 weeks of age. The lower body weight of +/+ female sat 26 weeks probably reflected the consequences of a slightly younger age at weaning, rather than a contribution of the ob mutation expressing in heterozygous state in 2/3 of the +/? females. Since there is some variation in the times at which ob/ob mice and their lean controls are weaned, investigators receiving mutant mice should determine body weights of the mice upon receipt, and use these initial weights as an internal control to assess interexperimental variation between separate shipments of mutant mice.

Figure 1. Body weight changes in C57BL/6J-ob/ob mice in comparison to two types of lean controls.

Based upon previously published studies using B6-ob/ob mice, we anticipated that B6-ob/ob mice would exhibit non-fasting hyperglycemia at 6 weeks of age, maintain elevated plasma glucose levels through puberty, and exhibit a remission from hyperglycemia (plasma glucose <250 mg/dl) somewhere between 12 and 16 weeks of age. As noted in Table 1 below, there was heterogeneity in the hyperglycemic syndrome produced in males, with male #1 remaining more resistant than the other 4 males. This male, although comparably obese to the other 4 at 4 weeks of age, was the least obese of the group at 26 weeks of age (59.6 grams compared to the group mean of 68.0±2.6 grams). In contrast, male #5 maintained a rather severe hyperglycemia through 18 weeks of age. Histopathological analysis of pancreases showed islet hyperplasia and beta cell hypertrophy in all ob/ob males in this group.

Table 1. Age-associated Changes in Plasma Glucose in Males
Genotype (n) ob/ob	Mouse # 1 2 3 4 5	6 wk 260 422 430 401 480	10 wk 210 264 415 466 538	12 wk 201 269 228 336 345	16 wk 157 158 241 332 466	18 wk 141 207 237 245 346	24 wk 232 216 207 259 279
mean ± sem		399 ± 37	379 ± 62	276 ± 29	271 ± 58	235 ± 33	230 ± 13
+/? (5) mean ± sem		224 ± 21	196 ± 16	212 ± 11	190 ± 12	205 ± 9	170 ± 8
+/+ (4) mean ± sem		214 ± 9	195 ± 11	197 ± 10	188 ± 6	197 ± 10	169 ± 8

Female plasma glucose values in Table 2 show that 4/5 of the B6-ob/ob females underwent remission from hyperglycemia by 16 weeks of age. Most of the variance in this group was contributed by ob/ob female #4, who exhibited the most severe hyperglycemia at 6 weeks of age, and remained chronically hyperglycemic throughout the study. Histological analysis showed a pattern of islet atrophy and pancreatic beta cell necrosis in the pancreas of this female, contrasting with the compensatory islet hyperplasia and beta cell hypertrophy typical of the other 4 mutant females. In contrast to the continued gain in body weight exhibited by the other 4 mutant females, female #4 reached its maximum weight of 49.4 grams at 8 weeks of age and then declined to 29.8 grams at 26 weeks. This contrasts to a mean weight of 64.6 grams for the other 4 females at 26 weeks. This inability to stabilize and then reverse hyperglycemia is characteristic of C57BL/KsJ-db/db (diabetes; 000642) mice of both sexes, but remains atypical of B6-ob/ob mice. This basis for this variation remains to be elucidated.

Table 2. Age-associated Changes in Plasma Glucose in Females
Genotype (n) ob/ob	Mouse # 1 2 3 4 5	6 wk 233 374 301 559 344	10 wk 183 301 278 704 247	12 wk 189 330 296 712 269	16 wk 166 184 194 692 185	18 wk 127 148 257 670 286	24 wk 144 167 172 556 211
mean ± sem		362 ± 55	343± 93	359 ± 91	284 ± 102	298 ± 98	250 ± 77
+/? (5) mean ± sem		204 ± 12	186 ± 10	183 ± 6	161 ± 7	164 ± 9	186 ± 11
+/+ (5) mean ± sem		204 ± 14	189 ± 9	203 ± 11	182 ± 10	163 ± 5	176 ± 12

Discussion

This limited survey of glycemic changes in B6-ob/ob mice indicates that while the period of hyperglycemia is transient, individual variability exists in both the degree of hyperglycemia attained, as well as the rate of remission from hyperglycemia. Given the variability among individuals, these are several things the investigator receiving B6-ob/ob mice from the Jackson Laboratory can do to limit variability. Most investigators prefer to withdraw blood from conscious mice since certain anethestics have been shown to artificially depress plasma glucose levels. Plasm glucose in B6-ob/ob mice has been shown to be particularly responsive to handling stress (Surwit et al. 1984).   Hence, it is important that (1) the mice be allowed to acclimate to the investigator's environment and receive some handling before blood samples are taken, (2) an experienced technician capable of sampling blood within 10 seconds of grasping the mouse should be employed and (3) the blood samplings should always be done at the same time of day since there are diurnal fluctuations. If experimental diets are utilized, a control group fed a natural ingredient chow diet should be included, since B6-+/+ males develop obesity and moderate hyperglycemia when fed high fat, high simple sugar, low fiber diet (Surwit et al. 1988). Finally, in comparing their values with those reported in the literature, investigators should be aware that plasma glucose values in mice range up to 30% higher than the corresponding blood glucose determinations from the same mice.

References

1. Coleman DL. Obese and diabetes: two mutant genes causing diabetes-obesity syndromes in mice. Diabetologia 1978; 14:141-48.

2. Coleman DL. Diabetes-obesity syndromes in mice. Diabetes 1982; 31 (Suppl 1):1-6.

3. Bray GA, Fisler J, York DA. Neuroendocrine control of the development of obesity: understanding gained from studies of experimental animal models. Frontiers in Obesity 1990; 112:128-81.

4. Surwit RS, Feinglos MN, Livingston EG, Kuhn CM, McCubbin JA. Behavioral manipulation of the diabetic phenotypes in ob/ob mice. Diabetes 1984; 33:616-18.

5. Surwit RS, Kuhn CM, Cochrane C, McCubbin JA, Feinglos MN. Diet-induced type II diabetes in C57BL/6J mice. Diabetes 1988; 37:1163-67.