The obese gene is expressed in lean littermates of the genetically obese mouse (C57BL/6J ob/ob)

Edwin W. Haller1, Lorentz E. Wittmers Jr.1, Irina V. Haller1, and Ronald R. Regal2

Departments of 1 Medical and Molecular Physiology and of 2 Mathematics and Applied Statistics, University of Minnesota, Duluth, Minnesota 55812


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Some individuals of the mixed group of "lean" littermates (+/ob and +/+) of (C57BL/6J ob/ob) often suggest phenotypic characteristics of ob/ob animals. Therefore, it was of interest to determine whether expression of the ob gene had physiological significance in +/ob animals. Body weight (BW), fasting blood glucose (FBG), and body core temperature (Tr) were monitored between 62 and 364 days of age in +/+ and +/ob mice. Among females but not males, +/ob mice were heavier (P = 0.003) and FBG levels were greater (P = 0.04) than in +/+ animals. Comparison of Tr indicated differences suggesting falling Tr in +/ob but rising Tr in +/+ mice with age in males but not females. Multivariate analysis of variance yielded genotype effects for both males (P = 0.002) and females (P = 0.02). BW, FBG, and Tr alone were sufficient at the 75% level for genotypic characterization and separation of +/? animals as +/ob or +/+; clearly, expression of the ob gene in heterozygotes of the +/ob animal may make the mixed +/? group inappropriate as lean controls.

genetic obesity; ob gene product; phenotypic differentiation; ob/ob experimental controls


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE GENETICALLY OBESE MOUSE remains a popular animal model for studying aspects of obesity and abnormal carbohydrate metabolism, specifically hyperglycemia. In most approaches, a mixed group of lean littermates has been used as the lean control group; however, they are of unknown genetic background. At best, two-thirds of lean animals are heterozygous, carrying one obese gene (+/ob), and the remaining one-third are of the homozygous wild type (+/+). Although the ob gene is recessive, observations have suggested that it may be expressed partially in heterozygotes (8). Therefore, comparison of homozygous obese (ob/ob) mutants with groups of genetically mixed "lean" littermates (+/?) of the C57BL/6J strain could have profound effects on experimental results. Consequently, it was of interest to confirm by an alternative approach whether expression of the recessive ob gene was indeed fully excluded in the +/ob animal or whether it may influence the physiology of carriers. The present paper examines this question and poses the possibility of separating +/ob mice from the +/+ mice in the +/? group by comparing noninvasive physiological parameters of body weight (BW), fasting blood glucose (FBG), and body core temperature (Tr).


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and experiments. For purposes of the present study, heterozygous mutants (+/ob, derived from ovarian transplants) and wild-type (+/+) mice of the C57BL/6J strain were supplied by The Jackson Laboratory. All animals were maintained on an unrestricted diet of standard mouse chow (Purina Mouse Chow) and a 12:12-h light-dark cycle at 25 ± 1°C. Before all experiments, animals were fasted (water ad libitum) for 16-20 h. Data obtained from 116 mice of the C57BL/6J strain were analyzed. Among males this analysis included 42 +/+ and 33 +/ob genotypes. Among females were included 25 +/+ and 16 +/ob genotypes. Each animal provided a single set of measurements of BW, FBG, and Tr. Individual animals were not monitored chronologically; however, the animals' ages ranged from 62 to 364 days. The analyses of physiological variables employed (see Statistical analyses) in this study were adjusted for age and took into account gender differences. Because this study involved comparison between +/+ and +/ob genotypes at various ages, only animals were used whose ages fell within ±20% of those in the +/ob test group.

Under light diethyl ether anesthesia, animals were weighed and core (rectal) body temperatures were measured in a thermoneutral environment (room temperature 25 ± 0.2°C with animals protected from draft) with a miniature thermistor (Bailey Instrument). Approximately 75 µl of blood were drawn from the retroorbital plexus for determination of blood glucose concentration by the glucose oxidase method (Glucose Analyzer model 23A, Yellow Springs Instrument).

Statistical analyses. The three physiological variables of BW, FBG, and Tr were analyzed separately for each gender by use of a general linear model that included potential effects of age and genotype and checking for outliers and needed transformations (24, 26). For purposes of multivariate analysis of variance and discriminant analysis, the variables were age-adjusted to a common age of 300 days. Genotype differences were tested by multivariate analysis of variance (26). Discriminant analysis (20) was performed to determine how well a mouse of unknown genotype could be classified. All statistical computations were done with SAS (22).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

For purposes of statistical analysis, it was desirable to consider a general model of linear functions for genotype and age in which the linear dependence of each variable could be demonstrated. In the present study, observations ranged over a considerable age span with unequal distribution of animals, resulting in asymmetry of the distribution of data for the three variables tested. To proceed with the necessary statistical analyses required first logarithmic data transformation for BW and FBG but not for Tr. Scatter plots were obtained demonstrating linear relationships of all variables relative to age (Figs. 1-3). For all cases except male Tr values, general linear model relationships to log(age) were parallel. For the purpose of applying discriminant analyses, the physiological values were adjusted to a common age of 300 days by use of the common slope. (The choice of 300 days was arbitrary and without effect on subsequent results.) However, for males the age effect on Tr was significantly different in the +/+ and +/ob genotypes [log(age) vs. (genotype) interaction P = 0.0002]. Because the aim of discriminant analysis was to predict the genotype of unknown cases, the Tr for males could not be adjusted separately for the two genotypes. Therefore, the data for Tr from both groups were combined to determine a common age adjustment. Alternatively, data analysis of the monitored variables might have been approached by grouping of genotype responses according to age; however, this option was rejected, because all variables changed continuously with age.


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Fig. 1.   Scattergram of individual data for body weight (BW) vs. age of male (A) and female (B) +/+ and +/ob mice of the C57BL/6J strain. The log(BW) vs. log(age) interaction was statistically significant for males (P = 0.0001) and for females (P = 0.0004). d, Days.


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Fig. 2.   Scattergram of individual data of fasting blood glucose (FBG) levels vs. age of male (A) and female (B) +/+ and +/ob mice of the C57BL/6J strain. The log(FBG) vs. log(age) interaction was statistically significant for males (P = 0.0024) and for females (P = 0.0001).


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Fig. 3.   Scattergram of individual data of core body temperature (Tr) vs. age of male (A) and female (B) +/+ and +/ob mice of the C57BL/6J strain. The log(Tr) vs. log(age) interaction was statistically significant for females (P = 0.0005).

Analysis of individual variables within gender and between genotypes failed to show differences in BW of males (Fig. 1A); however, female +/ob mice were heavier (P = 0.003) than their +/+ comparison groups (Fig. 1B). Similarly, whereas no differences were detected in the FBG levels of males (Fig. 2A), those of females were greater (P = 0.04) in +/ob than in +/+ animals (Fig. 2B). Finally, comparison of Tr indicated statistically significant differences between genotypes for males but not females, and Tr of males younger than 100 days of age appeared to be lower in +/+ than in +/ob mice. Regressions of Tr for males tended toward a positive slope for +/+ but a negative slope for +/ob mice relative to age (Fig. 3A); those for females appeared to be equivalent (Fig. 3B).

Multivariate analysis of variance for age-adjusted values of all three variables yielded significant overall genotype effects at the level of P = 0.002 for males and P = 0.02 for females.

Because of observed gender differences, discriminant analysis was performed separately for each gender by using the data of the three monitored physiological variables. Genotypic categories were defined by determination of the canonical variables as linear combinations of the physiological parameters or their transforms, i.e., logarithmic transforms of BW and FBG and of the raw data for Tr. Table 1 reflects the genotypic classification of animals by comparison of known genotype vs. genotype assigned on the basis of discriminant analysis. Correct identification was achieved for ~73% of +/+ and +/ob mice of either gender. Chance alone would have resulted in correct identification of genotypes at a level of 50%.

                              
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Table 1.   Discriminant analysis for both genders based on body weight, blood glucose concentration, and core temperature adjusted to age


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present studies show clear expression of the ob gene in the C57BL/6J +/ob heterozygous mouse of either gender compared with the +/+ wild type. This gene penetration is evident more strongly in female than in male animals in comparisons of the three individual physiological variables BW, FBG, and Tr. As indicated by multivariate analysis of variance, when the combined effect of all three variables was examined, penetration of the ob gene was clearly demonstrated in heterozygotes of either gender.

Although no influence could be demonstrated in males on the basis of linear regressions of age-adjusted data, multivariate analysis of variance illustrated a clear difference between males of the +/ob and +/+ genotypes. These results suggest the possibility of a relatively complex mode of expression of the ob gene product in males. In contrast, comparisons in females clearly illustrated statistical significance in genotype dependence of two of the three variables, BW and FBG, in their respective linear regressions. Although such results do not constitute proof of sex linkage of the ob gene, the possibility of such a relationship cannot be ignored. Thus our data support the hypothesis that the +/ob mouse differs significantly in certain physiological characteristics from the +/+ mouse, and these two animals should not be considered as equivalent.

Results from our study are important to several experimental approaches involving the genetically obese mouse model for examination of the obese-hyperglycemic syndrome (2, 11, 25) or for the purpose of investigating aspects of carbohydrate and fat metabolism (3, 4, 16), for which precise mechanisms remain to be elucidated (1, 20).

One of the most striking characteristics of the ob/ob animal is its extreme sensitivity to stress as manifested in the magnitude of hyperglycemia achieved by seemingly innocuous stimuli (9, 12). Both the adrenal medulla (10) and cortex (13, 23, 25) have been implicated as mediators of this response. Whereas the magnitude of this hyperglycemic response to stress may be so great that differences between +/+ and +/ob animals are obscured, that may not be the case in other situations when more subtle differences between lean and obese animals come into play. Such may be the case regarding the effect of the ob gene on the sensitivity of peripheral insulin receptors (2, 5, 13, 16).

As indicated above and confirmed recently (7), the ob gene affects adrenal mechanisms involved in stress responses. A recent abstract (19) claimed that no differences existed between +/+ and +/ob premature and postpubertal mice either in blood hormone levels, including that of corticosterone, or in their metabolic rates. Unfortunately, gender was not specified in this (19) study, and the study was terminated with postpubertal animals rather than extending to older mice. However, data presented here indicate a clear gender effect on the ob gene product, and this effect included much older, adult animals; these factors may account for the differences in results observed in the two studies.

Hypothermia (6) and compromised thermogenic capacity (21) are characteristics of the homozygote ob/ob mouse that were not clearly apparent in the present study in the +/ob heterozygotes of either gender. Only sketchy data are available in the literature for food consumption and metabolic rates of identified +/ob mice, over a narrow age range, and these data were not correlated with core body temperature (19). In light of published data concerning the regulation of food intake and lipid utilization involving the ob gene product leptin (18), the potential function of the ob gene on regulation of body core temperature should be examined further.

Discriminant analysis of the present data was an attempt to develop a tool for classification of genotypes of C57BL/6J mice on the basis of phenotypic expression of the ob gene. Multivariate analysis of variance that used the three parameters of BW, FBG, and Tr suggested that it might be possible to achieve such separation of mixed lean littermates, i.e, +/+ and +/ob, from the +/? group. That goal was partially reached, in that nearly three-fourths of both +/+ and +/ob animals of either gender were identified correctly by discriminant analysis when only BW, FBG, and Tr were used. This level of success strongly indicates that considerable expression of the ob gene exists in the +/ob heterozygote. Analysis of additional physiological parameters in +/ob mice may well lead to a statistical model that permits genotypic separation of these mice by noninvasive means.

Previous investigations have depended primarily on comparisons between ob/ob homozygotes and genetically unknown mixtures of +/ob heterozygotes and +/+ wild types. With the expression of the ob gene in +/ob heterozygotes, as our data suggest, the results from such experiments might have been affected by the ratio of +/ob to +/+ mice in the +/? pool of "control" animals. More precise estimation of the physiological effect of the ob gene should be possible by the exclusive use of +/+ mice as a comparison group.


    ACKNOWLEDGEMENTS

This work was presented in abstract form at the 23rd International Congress of Physiology, St. Petersburg, Russia, in 1997.


    FOOTNOTES

The study was supported in part by National Institute of General Medical Sciences Grant R5-GM-53421A and research funds from the Dept. of Medical and Molecular Physiology, School of Medicine, University of Minnesota.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: E. W. Haller, Medical and Molecular Physiology, Univ. of Minnesota, Duluth, MN 55812-2487 (E-mail: ehaller{at}d.umn.edu).

Received 4 September 1998; accepted in final form 5 January 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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3.   Connolly, E., and J. A. Carnie. Possible expression of the obese gene in the brown adipose tissue of lean heterozygote littermates of the genetically obese (ob/ob) mouse. Int. J. Obes. 8: 441-450, 1984[Medline].

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5.   Dubuc, P. U. Transient postweaning expression of excessive fat deposition and diabetes mellitus in ob/ob mice. Growth Dev. Aging 60: 145-151, 1996[Medline].

6.   Dubuc, P. U., N. J. Wilden, and H. J. Carlisle. Fed and fasting thermoregulation in ob/ob mice. Ann. Nutr. Metab. 29: 358-365, 1985[Medline].

7.   Feldkircher, K. M., A. M. Mistry, and D. R. Romsos. Adrenalectomy reverses pre-existing obesity in adult genetically obese (ob/ob) mice. Int. J. Nutr. 20: 232-235, 1996.

8.   Haller, E. W., and L. E. Wittmers. Sensitivity of the obese (ob/ob) mouse to exogenous insulin (Abstract). Endocrinology 104: 170A, 1979.

9.   Haller, E. W., and L. E. Wittmers. Expression of the obese gene in lean littermates of the genetically obese mouse (C57 BL/6J-ob/ob). FASEB J. 5: A607, 1993.

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Am J Physiol Endocrinol Metab 276(4):E762-E765
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




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